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DR.  WILLIAM  J.  OILS 

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in  Biolosical  Chemistry 


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PRACTICAL 
PHYSIOLOGICAL  CHEMISTRY 

HAWK 


Absorption  Spectra. 


Oxyhaemoglobin. 


Haemogflobin. 


Carboxy- 
haemog;lObln. 


Neutral  Met- 
haemoglobin. 


Alkaline  Met- 
haemoglobln. 


Alkali 
Haematln. 


Absorption  Spectra. 


s  % 


Reduced  Alkali 
Haematin  or 
Haemochromogen. 


Acid  Haematin  in 
ethereal  solution. 


Acid  Haemato- 
porphyrin. 


Alkaline 

Haematopor- 

phyrin. 


Urobilin  or  Hydro-' 
bilirubin  in  acid 
solution. 


Urobilin  or  Hydro- 
bilirubin  -in  alkalin 
solution  after  the 
addition  of  zinc 
chloride  solution. 


Bilicyanin  or 
Cholecyanin  in 
alkaline  solution. 


PRACTICAL  ~^ 

PHYSIOLOGICAL  CHEMISTRY 


A  BOOK  DESIGNED  FOR  USE  IN  COURSES  IN  PRACTICAL 

PHYSIOLOGICAL  CHEMISTRY  IN  SCHOOLS 

OF  MEDICINE  AND  OF  SCIENCE 


BY 
PHILIP  B.  HAWK,  M.  S.,  Ph.  D. 

PROFESSOR   OF   PHYSIOLOGICAL   CHEMISTRY   IN   THE   UNIVERSITY   OF   ILLINOIS 


THIRD  EDITION,  REVISED  AND  ENLARGED 


WITH  TWO  FULL-PAGE  PLATES  OF  ABSORPTION  SPECTRA  IN  COLORS, 

FOUR  ADDITIONAL  FULL-PAGE    COLOR    PLATES  AND  ONE 

HUNDRED  AND  TWENTY-SEVEN  FIGURES  OF  WHICH 

TWELVE  ARE  IN  COLORS 


PHILADELPHIA 

P.  BLAKISTON'S  SON  &  CO. 

1012  WALNUT  STREET 
1910 


First  Edition,  Copyright  iqoj,  by  P.  Blakiston's  Son  &  Co. 
Second  Edition,  Copyright  igog,  by  P.  Blakiston's  Son  &  Co. 
Third  Edition,  Copyright  igio,  by  P.  Blakiston's  Son  &  Co. 


H 


f'rhilrd  by 

'I  he  Maple  Press 

York,  Pa. 


THESE   PAGES    ARE 

AFFECTIONATELY     DEDICATED 

TO 

MY   MOTHER' 


PREFACE  TO  THIRD  EDITION 


The  increasing  approval  with  which  this  volume  is  being  received 
has  rendered  necessary  the  preparation  of  a  new  edition,  although  the 
period  elapsing  since  the  last  edition  appeared  is  little  more  than  one 
year.  The  present  edition  has  been  brought  up  to  date  by  the  inser- 
tion of  various  additions  and  corrections  as  well  as  by  the  inclusion  of 
a  number  of  qualitative  tests  and  quantitative  methods.  Because  of 
the  very  short  intervening  period  since  the  last  edition  of  the  volume, 
the  new  material  inserted  is  rather  small  in  quantity  when  compared 
with  that  incorporated  at  the  pre\ious  revision. 

The  author  wishes  to  thank  Dr.  W.  H.  Welker  and  Dr.  Croll  for 
permission  to  insert  unpublished  material. 
Urbana,  September,  1910. 


PREFACE  TO  SECOND  EDITION 


.  The  kind  reception  accorded  this  volume  by  the  instructors  in 
physiological  chemistry  in  the  United  States  and  Great  Britain  has 
made  the  preparation  of  a  new  edition  imperative,  notwithstanding 
the  fact  that  less  than  two  years  have  elapsed  since  the  former  edition 
appeared.  The  advance  and  development  made  in  the  field  of  physio- 
logical chemistry  during  this  period  have  been  both  rapid  and  impor- 
tant; conditions  which  would  of  themselves  have  necessitated  the  revision 
of  the  volume  at  an  early  date. 

The  book  has  been  thoroughly  revised  in  all  departments  and  in 
part  rewritten,  the  system  of  spelling  officially  adopted  by  the  American 
Chemical  Society  having  been  followed  throughout  the  volume. 
Besides  introducing  many  new  qualitative  tests  and  quantitative 
methods,  the  author  has  added  a  chapter  on  "Enzymes  and  Their 
Action"  and  has  rewritten  the  two  chapters  on  Proteins.  The  term 
"protein"  has  been  substituted  for  "proteid"  and  the  classification 
of  proteins  as  recently  adopted  by  the  American  Physiological  Society 
and  the  American  Society  of  Biological  Chemists  has  been  introduced 
and  is  followed  throughout  the  text;  the  classification  adopted  by  the 
Chemical  and  Physiological  Societies  of  England  is  also  included. 

The  original  plan  of  the  book  has  been  adhered  to  with  the  excep- 
tion that  the  chapter  on  "Enzymes  and  Their  Action"  has  been  made 
Chapter  I  and  the  practical  work  upon  the  proteins  is  preceded  by  a 
chapter  giving  a  brief  discussion  of  protein  substances  from  the  stand- 
point of  their  decomposition  and  synthesis.  We  believe  that  the  stu- 
dent will  be  able  to  pursue  his  practical  work  more  intelligently  and 
will  derive  greater  benefit  therefrom  if  the  plan  of  instruction  as  sug- 
gested in  Chapters  IV  and  V  be  followed  in  the  presentation  of  the 
subject  of  "Proteins." 

The  author  wishes  to  express  his  thanks  to  all  those  who  so  kindly 
offered  suggestions  for  the  betterment  of  the  book.  He  is  particularly 
desirous  of  expressing  his  gratitude  to  Professor  Lafayette  B.  Mendel 
and  Dr.  Thomas  B.  Osborne  for  the  many  helpful  suggestions  they 
have  so  kindly  given  him.  His  thanks  are  also  due  Professor  C.  A. 
Herter,  Dr.  H.  D.  Dakin,  Dr.  S.  R.  Benedict,  and  Mr.  S.  C.  Clark 

ix 


X  PREFACE    TO    SECOND    EDITION 

for  permission  to  insert  unpublished  material,  to  Mr.  Paul  E.  Howe 
for  valuable  assistance  rendered  in  the  reading  of  proof  and  in  the 
verification  of  tests  and  methods,  and  to  Dr.  M.  E.  Rehfuss  for  assist- 
ance in  proof  reading. 

The  author  takes  this  opportunity  of  making  an  acknowledg- 
ment which  was  inadvertently  omitted  from  the  first  edition.  He 
wishes  to  express  his  obligation  to  the  laboratories  of  physiological 
chemistry  at  Yale  University  and  at  Columbia  University  (College 
of  Physicians  and  Surgeons)  in  the  latter  of  which  he  was  Assistant 
to  Professor  W.  J.  Gies  for  two  years.  The  courses  given  in  these 
laboratories  formed  the  basis  of  many  of  the  experiments  included  in 
this  volume,  and  it  is  with  feeHngs  of  deepest  gratitude  that  he  records 
this  acknowledgement  of  the  assistance  thus  rendered  by  those  in 
charge  of  these  courses. 

Philip  B.  Hawk. 

Urbaxa,  Illinois, 
February,  1909. 


PREFACE  TO  FIRST  EDITION 


The  plan  followed  in  the  presentation  of  the  subject  of  this  volume 
is  rather  different,  so  far  as  the  author  is  aware,  from  that  set  forth 
in  any  similar  volume.  This  plan,  however,  he  feels  to  be  a  logical 
one  and  has  followed  it  with  satisfactory  results  during  a  period  of 
three  years  in  his  own  classes  at  the  University  of  Pennsylvania.  The 
main  point  in  which  the  plan  of  the  author  differs  from  those  previously 
proposed  is  in  the  treatment  of  the  food  stuffs  and  their  digestion. 

In  Chapter  IV  the  "Decomposition  Products  of  Proteids"  has 
been  treated  although  it  is  impracticable  to  include  the  study  of  this 
topic  in  the  ordinary  course  in  practical  physiological  chemistry. 
For  the  specimens  of  the  decomposition  products,  the  crystalline 
forms  of  which  are  reproduced  by  original  drawings  or  by  micro- 
photographs,  the  author  is  indebted  to  Dr.  Thomas  B.  Osborne  of 
New  Haven,  Conn. 

Because  of  the  increasing  importance  attached  to  the  examination 
of  feces  for  purposes  of  diagnosis,  the  author  has  devoted  a  chapter 
to  this  subject.  He  feels  that  a  careful  study  of  this  topic  deserves 
to  be  included  in  the  courses  in  practical  physiological  chemistry,  of 
medical  schools  in  particular.  The  subject  of  solid  tissues  (Chapters 
XIII,  XIV  and  XV)  has  also  been  somewhat  more  fully  treated  than 
has  generally  been  customary  in  books  of  this  character. 

The  author  is  deeply  indebted  to  Professor  Lafayette  B.  Mendel, 
of  Yale  University,  for  his  careful  criticism  of  the  manuscript  and  to 
Professor  John  Marshall,  of  the  University  of  Pennsylvania,  for  his 
painstaking  revision  of  the  proof.  He  also  wishes  to  express  his  grati- 
tude to  Dr.  David  L.  Edsall  for  his  criticism  of  the  clinical  portion  of 
the  volume;  to  Dr.  Otto  Folin  for  suggestions  regarding  several  of  his 
quantitative  methods,  and  to  Mr.  John  T.  Thomson  for  assistance  in 
proof  reading. 

For  the  micro-photographs  of  oxyhaemoglobin  and  haemin  repro- 
duced in  Chapter  XI  the  author  is  indebted  to  Professor  E.  T.  Reichert, 
of  the  University  of  Pennsylvania,  who,  in  collaboration  with  Professor 
A.  P.  Brown,  of  the  University  of  Pennsylvania,  is  making  a  very  ex- 

xi 


XU  PREFACE    TO    FIRST    EDITION 

tended  investigation  into  the  crystalline  forms  of  biochemic  substances. 
The  micro-photograph  of  allantoin  was  kindly  furnished  by  Professor 
Mendel.  The  author  is  also  indebted  for  suggestions  and  assistance 
received  from  the  lectures  and  published  writings  of  numerous  authors 
and  investigators. 

The  original  drawings  of  the  volume  were  made  by  Mr.  Louis 
Schmidt  whose  eminently  satisfactory  efforts  are  highly  appreciated 
by  the  author. 

Philip  B.  Hawk. 

Philadelphia. 


CONTENTS 


CHAPTER  I. 

Enzymes  and  Their  Action i 

CHAPTER  II. 
Carbohydrates 20 

CHAPTER  III. 

Salivary  Digestion 52 

CHAPTER  IV. 

Proteins:  Their  Decomposition  and  Synthesis 60 

CHAPTER  V. 

Proteins:  Their  Classification  and  Properties 83 

CHAPTER  VI. 

Gastric  Digestion 115 

CHAPTER  VII. 

Fats ^[28 

CHAPTER  VIII. 

Pancreatic  Digestion 137 

CHAPTER  IX. 
Bile 147 

CHAPTER  X. 

Putrefaction  Products 158 

CHAPTER  XI. 

Feces 168 

CHAPTER  XII. 

Blood ; 178 

xiii 


XIV  CONTEXTS 

CHAPTER  XIII. 
Milk 213 

CHAPTER  XIV. 
Epithell\l  and  Connective  Tissues 223 

CHAPTER  XV. 
MUSCUL.A.R  Tissue 231 

CHAPTER  XVI. 

Nervous  Tissue 244 

CHAPTER  XVII. 
Urine:  General  Characteristics  of  Normal  and  Pathological 

Urine 250 

CHAPTER  XVIII. 

Urine:  Physiological  Constituents 259 

CHAPTER  XIX. 

Urine:  Pathological  Constituents 299 

CHAPTER  XX. 
Urine:  Organized  and  Unorganized  Sediments 338 

CHAPTER  XXI. 

Urine:  Calculi 357 

CHAPTER  XXII. 

Urine:  Quantitative  Analysis 361 

CHAPTER  XXIII. 

Quantitative  Analysis  ok  Milk,  Gastric  Juice  and  Blood 404 

Appendix 411 

Index 421 


LIST  OF  ILLUSTRATIONS 


Plate 

I.  Absorption  Spectra  1                                                                  „        .  . 

^^     . ,  .       ^  > tronUspiece 

II.  Absorption  Spectra  J 

III.  Osazones Opposite  page  23 

IV.  Normal  Erythrocytes  and  Leucocytes Opposite  page  180 

V.  Uric  Acid  Crystals Opposite  page  267 

VI.  Ammonium  Urate Opposite  page  343 

Figure  Page 

1.  Dialyzing  Apparatus  for  Students'  Use 25 

2.  Einhorn  Saccharometer 31 

3.  One  Form  of  Laurent  Polariscope 32 

4.  Diagrammatic  Representation  of  the  Course  of  the  Light  through 

the  Laurent  Polariscope 33 

5.  Polariscope  (Schmidt  and  Hansch  Model) 33 

6.  Iodoform 42 

7.  Potato  Starch 44 

8.  Bean  Starch 44 

9.  Arrowroot  Starch 44 

10.  Rye  Starch 44 

11.  Barley  Starch 44 

12.  Oat  Starch •. 44 

13.  Buckwheat  Starch 44 

14.  Maize  Starch 44 

15.  Rice  Starch 44 

16.  Pea  Starch 44 

17.  Wheat  Starch 44 

18.  Microscopical  Constituents  of  Saliva 55 

19.  Glycocoll  Ester  Hydrochloride 67 

20.  Serine 68 

21.  Phenylalanine 69 

22.  Fischer  Apparatus 70 

23.  Tyrosine 71 

24.  Cystine 71 

25.  Histidine  Bichloride 73 

26.  Leucine 75 

27.  Lysine  Picrate 76 

XV 


XVI  LIST    OF    ILLUSTRATIONS 

Figure  Page 

28.  Aspartic  Acid 77 

29.  Glutamic  Acid 78 

30.  Laevo-a-Proline 79 

31.  Copper  Salt  of  Proline 79 

7,2.  Coagulation  Temperature  Apparatus 98 

T,T,.  Edestin loi 

34.  Excelsin,  the  Protein  of  the  Brazil  Nut 103 

35.  Beef  Fat 128 

36.  Mutton  Fat 131 

37.  Pork  Fat 133 

38.  Palmitic  Acid 134 

39.  Melting-Point  Api)aratus 135 

40.  Bile  Salts 149 

41.  Bilirubin  (Haematoidin) .' 150 

42.  Cholesterol 155 

43.  Taurine 156 

44.  Glycocoll 157 

45.  Ammonium  Chloride 162 

46.  Microscopical  Constituents  of  Feces 168 

47.  Ha^matoidin  Crystals  from  Acholic  Stools 169 

48.  Charcot-Leyden  Crystals 170 

49.  Boas'  Sieve 172 

50.  Oxyhaemoglobin  Crystals  from  Blood  of  the  Guinea  Pig 182 

51.  Gxyhx'moglobin  Crystals  from  Blood  of  the  Rat 182 

52.  Gxyhtemoglofjin  Crystals  from  Blood  of  the  Horse 183 

53.  Oxyhiemoglobin  Crystals  from  Blood  of  the  Squirrel 183 

54.  Oxyhcemoglobin  Crystals  "from  Blood  of  the  Dog 184 

55.  Oxyhcemoglobin   Crystals  from  Blood  of  the  Cat 184 

56.  Oxyha,'moglobin  Crystals  from  Blood  of  the  Necturus 185 

57.  Effect  of  Water  on  Erythrocytes 191 

58.  Hiemin  Crystals  from  Human  Blood 194 

59.  Hiemin  Crystals  from  Sheep  Blood 194 

60.  Sodium  Chloride 196 

61.  Direct-vision  Spectrosco|)e 199 

62.  Angular-vision  Spectroscope  Arranged  for  Absorjjlion  Analysis.  .  .  200 

63.  Diagram  of  Angular-vision  Spi(  irosc ope 200 

64.  Fleischl's  H;emometer 203 

65.  Pij>ctte  of  Fleischl's  Ha.'momeler. 204 

66.  Colored  (ilass  Weflge  of  Fleis(  hl's  Hamomeler 204 

67.  Dare's  Ila-moglobinometer 206 

68.  Horizontal  Se(  lion  of  Dare's  [|;iinoglol)inonieler 207 

69.  Method    of    i'illing    the    Capillary    Observation    Cell    of     Dare's 

H.'emoglobinometer 207 

70.  Thoma  Zeiks  Counting  ( "liamber 208 


LIST    OF    ILLUSTRATIONS  XVll 

Figure  Page 

71.  Thoma-Zeiss  Capillary  Pipettes 209 

72.  Ordinary  Ruling  of  Thoma-Zeiss  Counting  Chamber 210 

73.  Zappert's  Modified  Ruling  of  Thoma-Zeiss  Counting  Chamber.  .  211 

74.  Normal  Milk  and  Colostrum 214 

75.  Lactose 215 

76.  Calcium  Phosphate 220 

77.  Creatine 234 

78.  Xanthine 235 

79.  Hypoxanthine  Silver  Nitrate 241 

80.  Xanthine  Silver  Nitrate 242 

81.  Deposit  in  x\mmonical  Fermentation 253 

82.  Deposit  in  Acid  Fermentation 253 

83.  Urinometer  and  Cylinder 254 

84.  Beckmann-Heidenhain  Freezing-Point  Apparatus 256 

85.  Urea 261 

86.  Urea  Nitrate 263 

87.  Melting-Point  Tubes  Fastened  to  Bulb  of  Thermometer 264 

88.  Urea  Oxalate 265 

89.  Pure  Uric  Acid 269 

90.  Creatinine 271 

91.  Creatinine-Zinc  Chloride 272 

92.  Hippuric  Acid 276 

93.  Allantoin  from  Cat's  Urine 280 

94.  Benzoic  Acid 284 

95.  Calcium  Sulphate 292 

96.  "Triple  Phosphate" 296 

97.  The  Purdy  Electric  Centrifuge 338 

98.  Sediment  Tube  for  the  Purdy  Electric  Centrifuge 338 

99.  Calcium  Oxalate 340 

100.  Calcium  Carbonate 341 

loi.  Various  Forms  of  Uric  Acid 342 

102.  Acid  Sodium  Urate 343 

103.  Cystine 343 

104.  Crystals  of  Impure  Leucine 344 

105.  Epithelium  from  Different  Areas  of  the  Urinary  Tract 347 

106.  Pus  Corpuscles 348 

107.  Hyaline  Casts 349 

108.  Granular  Casts 350 

109.  Granular  Casts 351 

no.  Epithelial  Casts 351 

111.  Blood,  Pus,  Hyaline  and  Epithelial  Casts 351 

112.  Fatty  Casts 352 

113.  Fatty  and  Waxy  Casts 352 

114.  Cylindroids 353 


XVlll  LIST    OF   ILLUSTRATIONS 

Figure  Page 

115.  Crcnated  Erythrocytes 354 

116.  Human  Spermatozoa 355 

117.  Esbach's  Albuminometer 362 

118.  Marshall's  Urea  Apparatus 369 

119.  Hlifner's  Urea  Apparatus 371 

120.  Doremus- Hinds  Ureometer 372 

121.  Folin's  Urea  Apparatus 373 

122.  Folin's  Ammonia  Apparatus 374 

123.  Folin  Improved  Absorption  Tube 375 

124.  Berthelot-Atwater  Bomb  Calorimeter 382 

125.  CroU's  Fat  Apparatus 404 

126.  Soxhlet  Apparatus 405 

127.  Feser's  Lactoscope 406 


PHYSIOLOGICAL  CHEMISTRY. 


CHAPTER  I. 

ENZYMES  AND  THEIR  ACTION. 

According  to  the  old  classification  ferments  were  divided  into 
two  classes,  the  organized  ferments  and  the  unorganized  ferments.  As 
organized  ferments  or  true  ferments  there  were  grouped  such  sub- 
stances as  yeast  and  certain  bacteria  which  were  supposed  to  act  by 
virtue  of  vital  processes,  whereas  the  unorganized  ferments  included 
salivary  amylase  (ptyalin),  gastric  protease  (pepsin),  pancreatic  protease 
(trypsin),  etc.,  which  were  described  as  "non-living  unorganized  sub- 
stances of  a  chemical  nature."  Kiihne  designated  this  latter- class  of 
substances  as  enzymes  (ev  ^v^Liq — in  yeast).  This  division  into  organized 
ferments  (true  ferments)  and  unorganized  ferments  (enzymes)  was  gen- 
erally accepted  and  was  practically  unquestioned  until  Buchner  Over- 
threw it  in  the  year  1897  by  his  epoch-making  investigations  on  zymase. 
Previous  to  this  time  many  writers  had  expressed  the  opinion  that  the 
action  of  the  ferment  organisms  was  similar  to  that  of  the  unorganized 
ferments  or  enzymes  and  that  therefore  the  activity  of  the  former  was 
possibly  due  to  the  production  of  a  substance  in  the  cell,  which  was 
in  nature  similar  to  an  enzyme.  Investigation  after  investigation, 
however,  failed  to  isolate  any  such  principle  from  an  active  cell  and 
the  exponents  of  the  "vital"  theory  became  strengthened  in  their 
belief  that  certain  fermentative  processes  brought  about  by  living  cells 
could  not  occur  apart  from  the  biological  activity  of  such  cells.  How- 
ever, as  early  as  1858,  Traube  had  enunciated,  in  substance,  the  prin- 
ciples which  were  destined  to  be  fundamental  in  our  modern  theory 
of  fermentation.  He  expressed  the  belief  that  the  yeast  cell  produced 
a  product  in  its  metabolic  activities  which  had  the  property  of  reacting 
with  sugar  with  the  production  of  carbon  dioxide  and  alcohol,  and 
further  that  this  reaction  between  the  product  of  the  metabolism  of  the 
yeast  cell  and  the  sugar  occurred  without  aid  from  the  original  cell.  It 
was  not  until  1897,  however,  that  this  theory  was  placed  upon  a  firm 

I 


2  PHYSIOLOGICAL    CHEMISTRY. 

experimental  basis.  This  was  brought  about  through  the  efforts  of 
Buchner  who  succeeded  in  isolating  from  the  living  yeast  cells  a  sub- 
stance (zymase)  which,  when  freed  from  the  last  trace  of  organized 
cellular  material,  was  able  to  bring  about  the  identical  fermentative 
processes,  which,  up  to  this  time,  had  been  deemed  possible  only  in 
the  presence  of  the  active,  living  yeast  cell. 

Buchner's  manipulation  of  the  yeast  cells  consisted  in  first  grind- 
ing them  with  sand  and  infusorial  earth,  after  which  the  finely  divided 
material  was  subjected  to  great  pressure  (300  atmospheres)  and  yielded 
a  liquid  which  possessed  the  fermentative  activity  of  the  unchanged 
yeast  cell.^  This  liquid  contained  zymase,  the  principal  enzyme  of 
the  yeast  cell.  Later  the  lactic-acid-  and  acetic-acid-producing  bac- 
teria were  subjected  by  Buchner  to  treatment  similar  to  that  accorded 
the  yeast  cells,  and  the  active  intracellular  enzymes  were  obtained. 
Many  other  instances  are  on  record  in  which  a  soluble,  active  agent 
has  been  isolated  from  ferment  cells,  with  the  result  that  it  is  pretty 
well  established  that  all  the  so-called  organized  ferments  elaborate 
substances  of  this  character.  Therefore,  basing  our  definition  on  the 
work  of  Buchner  and  others  we  may  define  an  enzyme  as  an  unorgan- 
ized, soluble  ferment,  which  is  elaborated  by  an  animal  or  vegetable  cell 
and  whose  activity  is  entirely  independent  of  any  of  the  life  processes  of 
such  a  cell.  According  to  this  definition  the  enzyme  zymase  elaborated 
by  the  yeast  cell  is  entirely  comparable  to  the  enzyme  pepsin  elaborated 
by  the  cells  of  the  stomach  mucosa.  One  is  derived  from  a  vegetable 
cell,  the  other  from  an  animal  cell,  yet  the  activity  of  neither  is  depend- 
ent upon  the  integrity  of  the  cell. 

Enzymes  act  by  catalysis  and  hence  may  be  termed  catalyzers 
or  catalysts.  A  simple  rough  definition  of  a  catalyzer  is  "a  substance 
which  alters  the  velocity  of  a  chemical  reaction  without  undergoing 
any  apparent  physical  or  chemical  change  itself  and  without  becoming 
a  part  of  the  product  formed."  It  is  a  well-known  fact  that  the  velocity 
of  the  greater  number  of  chemical  reactions  may  be  changed  through 
the  presence  of  some  catalyzer.  For  example,  take  the  case  of  hydro- 
gen peroxide.  It  spontaneously  decomposes  slowly  into  water  and 
oxygen.  In  the  presence  of  colloidal  platinum,^  however,  the  decom- 
[)Osition  is  much  accelerated  and  ceases  only  when  the  destruction  of 
the  hydrogen  peroxide  is  complete.  Without  multiplying  instances, 
suffice  it  to  say  that  there  is  an  analogy  between  inorganic  catalyzers 

*  In  later  investigations  the  j^rocess  was  im|)rovef]  by  freezing  tlic  ground  (ells  will) 
lifjuid  air  and  finely  pulverizing  them  before  applying  the  |)ressurc, 

'•'  Pfftfiuccd  by  the  passage  of  electric  si>arks  between  two  plalinnin  Icriiiinals  inim(;rsc(i 
in  distilled  water,  thus  liberating  ultra-microscoijic  partic  les. 


ENZYMES   AND    THEIR  ACTION.  3 

and  enzymes,  the  main  point  of  difference  between  the  enzymes  and 
most  of  the  inorganic  catalyzers  being  that  the  enzymes  are  colloids.^ 

Inasmuch  as  each  of  the  enzymes  has  an  action  which  is  more  or  less 
specific  in  character,  and  since  it  is  a  fairly  simple  matter,  ordinarily,  to 
determine  the  character  of  that  action,  the  classification  of  the  enzymes 
is  not  attended  with  very  great  difficulties.  They  are  ordinarily  classi- 
fied according  to  the  nature  of  the  substrate^  or  according  to  the  type 
of  reaction  they  bring  about.  Thus  we  have  various  classes  of  enzymes, 
such  as  amylolytic,^  proteolytic,  lipolytic,  glycolytic,  uricolytic,  autolytic, 
oxidizing,  reducing,  inverting,  protein-coagulating,  deamidizing,  etc.  In 
every  instance  the  class  name  indicates  the  individual  type  of  enzy- 
matic activity  which  the  enzymes  included  in  that  class  are  capable  of 
accomplishing.  For  example,  amylolytic  enzymes  facilitate  the 
hydrolysis  of  starch  (amylum)  and  related  substances,  lipolytic  enzymes 
facilitate  the  hydrolysis  of  fats  (Aittos),  whereas  through  the  agency  of 
uricolytic  enzymes  uric  acid  is  broken  down.  There  is  a  tendency, 
at  the  present  time,  to  harmonize  the  nomenclature  of  the  enzymes  by 
the  use  of  the  termination,  -ase.  According  to  this  system  of  nomen- 
clature, all  starch-transforming  enzymes,  or  so-called  amylolytic 
enzymes,  are  called  amylases,  all  fat-splitting  enzymes  are  called  lipases, 
etc.  Thus  ptyalin  the  amylolytic  enzyme  of  the  saliva,  would  be 
termed  salivary  amylase  in  order  to  distinguish  it  from  pancreatic  amy- 
lase (amylopsin)  and  vegetable  amylases  (diastase,  etc.).  According 
to  the  same  system,  the  fat-splitting  enzyme  of  the  gastric  juice  would 
be  termed  gastric  lipase  to  differentiate  it  from  pancreatic  lipase  (steap- 
sin),  the  fat-splitting  enzyme  of  the  pancreatic  juice. 

Our  knowledge  regarding  the  distribution  of  enzymes  has  been 
wonderfully  broadened  in  recent  years.  Up  to  within  a  few  years, 
the  real  scientific  information  as  to  the  enzymes  of  the  animal  organism, 
for  example,  was  limited,  in  the  main,  to  a  rather  crude  understanding 
of  the  enzymes  intimately  connected  with  the  main  digestive  func- 
tions of  the  organism.  We  now  have  occasion  to  believe  that  enzymes 
are  doubtless  present  in  every  animal  cell  and  are  actively  associated 
with  all  vital  phenomena.  As  a  preeminent  example  of  such  cellular 
activity  may  be  cited  the  liver  cell  with  its  reputed  complement  of 
15-20  or  more  enzymes. 

*  Bredig  has  been  able  to  obtain  certain  inorganic  catalyzers  in  colloidal  solution. 
These  he  calls  "inorganic  enzymes." 

^  Substance  acted  upon. 

^ATmstrong  suggests  the  use  of  the  termination  "clastic"  instead  of  "lytic."  He 
calls  attention  to  the  fact  that  amylolytic,  in  analogy  with  electrolytic,  means  "decomposition 
by  means  of  starch"  and  is  therefore  a  misnomer.  He  suggests  the  use  of  amyloclastic, 
proteoclastic,  etc. 


4  PHYSIOLOGICAL    CHEMISTRY. 

In  text-book  discussions  of  the  enzymes  it  is  customary  to  say  that 
very  little  is  known  regarding  the  chemical  characteristics  of  these 
substances  since  no  member  of  the  enzyme  group  has,  up  to  the  present 
time,  been  prepared  in  an  absolutely  pure  eanditlon.  Apparently,  how- 
ever, from  the  nature  of  the  facts  in  the  case,  it  would  be  much  more 
accurate  to  say  that  we  absolutely  do  not  know  whether  a  specific  enzyme 
Jias,  or  has  not,  been  prepared  in  a  pure  state.  (Some  authors,  like 
Arthus,  have  assumed  that  enzymes  are  not  chemical  individuals,  but 
properties  conferred  upon  bodies.)  The  enzymes  are  very  difficult  to 
prepare  in  anything  like  a  condition  approximating  purity,  since  they 
are  very  prone  to  change  their  nature  during  the  process  by  which  the 
investigator  is  attempting  to  isolate  them.  For  this  reason  we  have 
absolutely  no  proof  that  the  final  product  obtained  is,  or  is  not,  in  the 
same  state  of  purity  it  possessed  in  the  original  cell.  Some  of  the  en- 
zymes are  more  or  less  closely  associated  with  the  proteins  from  the  fact 
that  they  are  both  formed  in  every  cell  as  the  result  of  the  cellular  ac- 
tivity, both  may  be  removed  from  solution  by  "salting-out,"  both  arc 
for  the  most  part  non-diffusible  and  are  probably  very  similar  as 
regards  elementary  composition.  Hence  in  the  preparation  of  some 
enzymes  it  is  extremely  difficult  to  make  an  absolute  separation  from 
the  protein.^  Under  certain  conditions  enzymes  are  readily  adsorbed 
by  shredded  protein  material,  such  as  fibrin,  and  may  successfully 
resist  the  most  prolonged  attempts  at  washing  them  free.  We  may 
summarize  some  of  the  properties  of  the  great  body  of  enzymes  as 
follows:  Enzymes  are  soluble  in  dilute  glycerol,  sodium  chloride  solu- 
tion, dilute  alcohol  and  water,  and  precipitable  by  ammonium  sulphate 
and  strong  alcohol.  Their  presence  may  be  proven  from  the  nature 
of  the  end-products  of  their  action  and  not  through  the  agency  of  any 
chemical  test.  They  arc  colloidal  and  non-diffusible ,  and  occur  closely 
associated  with  protein  material  with  which  they  ])ossess  many  proper- 
ties in  common.  Each  enzyme  shows  the  greatest  activity  at  a  certain 
temperature  called  the  optimum  temperature;  there  is  also  a  minimum 
and  a  maximum  temperature  for  each  specific  enzyme.  Their  action 
is  inhibited  by  sufficiently  lowering  the  temperature,  and  the  enzyme,  if 
in  solution,  is  entirely  destroyed  by  sul)jecting  it  to  a  temperature  of 
ioo°  C.  The  best  known  enzymes,  whether  derived  from  warm-blooded 
or  cold-blooded  animals,  are  most  active  between  35° -45'^  C.  The 
nature  of  the  surrounding  media  alters  the  velocity  of  the  enzymatic 
action,  some  enzymes  being  more  active  in  acid  sohition  whereas 
others  reriuire  an  alkaline  fluid. 

'  Others  seem  to  he  like  the  substrate  on  v\hi(  li  they  ;i(  t,  r.  _q.,  ( iirholiydrate. 


ENZYMES   AND    THEIR  ACTION.  5 

Many  of  the  more  important  enzymes  do  not  occur  preformed 
within  the  cell,  but  are  present  in  the  form  of  a  zymogen  or  mother- 
substance.  In  order  to  yield  the  active  enzyme  this  zymogen  must  be 
transformed  in  a  certain  specific  manner  and  by  a  certain  specific  sub- 
stance. This  transformation  of  the  inactive  zymogen  into  the  active 
enzyme  is  termed  activation.  For  instance,  the  zymogen  of  the  enzyme 
pepsin  of  the  gastric  juice,  termed  pepsinogen,  is  activated  by  the  hydro- 
chloric acid  secreted  by  the  gastric  cells  (see  p.  ii6),  whereas  the  acti- 
vation of  the  trypsinogen  of  the  pancreatic  juice  is  brought  about  by  a 
substance  termed  enterokinase^  (see  p.  138).  These  are  examples  of 
many  well-known  activation  processes  going  on  continually  within  the 
animal  organism.  The  agency  which  is  instrumental  in  activating  a 
zymogen  is  generally  termed  a  zymo-exciter  or  a  kinase.  In  the  cases 
cited  hydrochloric  acid  would  be  termed  a  zymo-exciter  and  entero- 
kinase  would  be  termed  a  kinase. 

After  filtering  yeast  juice,  prepared  by  the  Buchner  process  (see  p. 
2),  through  a  Martin  gelatin  filter,  Harden  and  Young  showed  that 
the  colloids  left  behind  and  the  filtrate  were  both  inactive  fermenta- 
tively.  Upon  treating  the  colloid  material  (enzyme)  with  some  of  the 
filtrate,  however,  the  mixture  was  shown  to  be  able  to  bring  about  pro- 
nounced fermentation.  It  is  believed  that  a  co-enzyme  present  in  the 
filtrate  was  the  efficient  agent  in  the  transformation  of  the  inactive 
enzyme.  It  is  necessary  to  make  frequent  renewals  of  the  co-enzyme 
in  order  to  maintain  continuous  fermentation.  It  was  further  shown 
that  this  co-enzyme,  in  addition  to  being  diffusible,  was  not  destroyed 
by  boiling  and  that  it  disappeared  from  yeast  juice  when  this  latter 
was  fermented  or  allowed  to  undergo  autolysis.  The  exact  nature  of 
this  co-enzyme  of  zymase  is  unknown.  The  co-enzyme  action,  in  this 
case,  is  probably  dependent  upon  the  presence  of  two  individual 
agencies,  one  of  which  is  phosphates. 

It  has  been  shown  by  Loevenhart  that  the  property  of  acting  as  a 
pancreatic  lipase  co-enzyme  is  vested  in  bile  salts,  and  Magnus  has 
further  shown  that  the  synthetic  salts  are  as  efficient  in  this  regard  as 
the  natural  ones.  A  few  other  instances  of  co-enzyme  demonstrations 
have  been  reported. 

The  so-called  "specificity"  of  enzyme  action  is  an  interesting  and 
important  fact.  That  enzymes  are  very  specific  as  to  the  character  of 
the  substrate,  or  substance  acted  upon,  is  well  known.  Emil  Fischer 
investigated  this  problem  of  specificity  extensively  in  connection  with 

'  Accordino;  to  Delezenne,  trypsinogen  may  be  rapidly  activated  by  soluble  calcium 
salts. 


6  PHYSIOLOGICAL    CHEMISTRY. 

the  fermentation  of  sugars  and  reached  the  conclusion  that  enzymes, 
with  the  possible  exception  of  certain  oxidases,  can  act  only  upon  such 
substances  as  have  a  specific  stereo-isomeric  relationship  to  themselves. 
He  considers  that  the  enzyme  and  its  substrate  must  have  an  inter- 
relation, such  as  the  key  has  to  the  lock,  or  the  reaction  does  not  occur. 
Fischer  was  able  to  predict,  in  certain  definite  cases,  from  a  knowledge 
of  the  constitution  and  stereo-chemical  relationships  of  a  substance, 
whether  or  not  it  would  be  acted  upon  by  a  certain  enzyme.  An 
application  of  this  specificity  of  enzyme  action  may  be  seen  in  the  well- 
known  facts  that  certain  enzymes  act  on  carbohydrates,  others  on  fats, 
and  others  on  protein;  and,  moreover,  that  the  group  of  those  which 
transform  carbohydrates,  for  example,  is  further  subdivided  into  spe- 
cific enzymes  each  of  which  has  the  power  of  acting  alone  upon  some 
one  sugar. 

It.  has  been  conclusively  shown,  in  the  case  of  certain  enzymes,^ 
at  least,  that  their  action  is  a  reversible  one  and  is,  in  all  its  main  fea- 
tures, directly  analogous  to  the  reversible  reactions  produced  by  chem- 
ical means.  For  instance,  in  the  saponification  of  ethyl-butyrate  by 
means  of  pancreatic  lipase,  it  has  been  shown  that  upon  the  formation 
of  the  end-products  of  the  reaction,  i.  e.,  butyric  acid  and  ethyl  alcohol, 
there  is  reversion'  and  the  reaction  is  stationary.  This  does  not 
mean  there  are  no  chemical  changes  going  on,  but  simply  indicates 
that  chemical  equilibrium  has  been  established,  and  that  the  change 
in  one  direction  is  counterbalanced  by  the  change  in  the  opposite 
direction.  Pancreatic  lipase  was  one  of  the  first  enzymes  to  have  the 
reversibility  of  its  reaction  clearly  demonstrated.^  A  knowledge  of 
the  fact  that  lipase  possesses  this  reversibility  of  action  is  of  extreme 
physiological  importance  and  aids  us  materially  in  the  explanation  of 
the  processes  involved  in  the  digestion,  absorption,  and  deposition  of 
fats  in  the  animal  organism  (see  p.  130). 

In  respect  to  many  enzymes  it  has  Ijcen  found  that  the  law  govern- 
ing the  action  of  inorganic  catalyzers  is  directly  applicable,  i.  e.,  thai 
the  intensity  is  almost  directly  proportional  to  the  concentration  of  the 
enzyme.  In  the  case  of  enzymes,  however,  there  is  a  difference  in  that 
a  maximum  intensity  is  soon  reached  and  that  subsequent  concentra- 
tion of  the  enzyme  is  productive  of  no  further  increase  in  intensity. 

'  This  is  probably  a  general  (:()n(lilif)n. 

*  The    re-synthesis   of   ethyl-hiityriitc    from    ils    liy<In)l\sis    ])ro(]iicts.     This   may    Ijc 
indicated  thus: 

Ethyl  InUyrate.  liulyric  acid.  lithyl  alcohol. 

'The  principle  was  first  dcmonstratf-d  in  ronnc(linn  \\'\\h  \hv  ch/aiiu-  maitasc  (sec 
P-  54). 


ENZYMES   AND    THEIR   ACTION.  7 

The  enzymes  which  have  been  shown,  to  obey  this  linear  law  are  lipase, 
invertase,  rennin,  and  trypsin.  In  certain  instances,  where  this  law  of 
direct  proportionality  between  the  intensity  of  action  and  the  concen- 
tration of  enzyme  does  not  hold,  it  has  been  found  that  the  law  of 
Schiltz,  first  experimentally  demonstrated  by  E.  Schiitz,  was  applicable. 
This  is  to  the  effect  that  the  intensity  is  directly  proportional  to  the 
square  root  of  the  concentration,  or  conversely,  that  the  relative  con- 
centrations of  enzymes  are  directly  proportional  to  the  squares  of  the 
intensities.  ^ 

It  has  been  shown  that  there  are  certain  substances  which  possess 
the  property  of  directly  inhibiting  or  preventing  the  action  of  a  catalyzer. 
These  are  called  anti-catalyzers  or  paralyzers  and  have  been  compared 
to  the  anti-toxins.  Related  to  this  class  of  anti-catalytic  agents  stand 
the  anti-enzymes.  The  first  anti-enzyme  to  be  reported  was  the  anii- 
rennin  of  Morgenroth.  This  was  produced  by  injecting  into  an  animal 
increasing  doses  of  rennet  solution,  whereupon  an  "anti"  substance 
was  subsequently  found  both  in  the  serum  and  in  the  milk,  which 
prevented  the  enzyme  rennin  from  exerting  its  normal  activity  in  the 
presence  of  caseinogen.  In  other  words,  anti-rennin  had  been  formed 
in  the  serum  of  the  animal,^  through  the  repeated  injections  of  rennet 
solution.  Since  the  discovery  of  this  anti-enzyme,  anti-bodies  have 
been  demonstrated  for  pepsin,  trypsin,  lipase,  urease,  amylase,  laccase, 
tyrosinase,  emulsin,  papain,  and  thrombin.  According  to  Weinland, 
the  reason  why  the  stomach  does  not  digest  itself  is,  that  during  life 
there  is  present  in  the  mucous  membrane  of  the  stomach  an  anti- 
enzyme  (anti-pepsin)  which  has  the  property  of  inhibiting  the  action  of 
pepsin.  A  similar  substance  (anti-trypsin)  is  present  in  the  intestinal 
mucosa  as  well  as  in  the  tissues  of  various  intestinal  worms.  Some 
investigators  are  not  inclined  to  accept  the  enzyme  nature  of  these 
inhibitory  agents  as  proven. 

EXPERIMENTS   ON   ENZYMES  AND   ANTI-ENZYMES. 

A.  Experiments  on  Enzymes.^ 

I.  AMYLASES. 

I.  Demonstration  of  Salivary  Amylase/ — To  25  c.c.  of  a  one 
per  cent  starch  paste  in  a  small  beaker,  add  5  drops  of  saliva  and  stir 

'  This  law  of  Schiitz  is  not  generally  applicable. 

-  Serum  is  normally  anti-tryptic. 

'  If  it  is  deemed  advisable  by  the  instructor  to  give  all  the  practical  work  upon  enzymes 
at  this  point  in  the  course,  additional  experiments  will  be  found  in  Chapters  III,  VI  and 
VIII. 

''  For  a  discussion  of  this  enzyme  see  p.  53. 


8  PHYSIOLOGICAL    CHEMISTRY. 

thoroughly.  At  intervals  of  a  minute  remove  a  drop  of  the  solution 
to  one  of  the  depressions  of  a  test-tablet  and  test  by  the  iodine  test.^ 
If  the  blue  color  with  iodine  still  forms  after  five  minutes,  add  another 
five  drops  of  saliva.  The  opalescence  of  the  starch  solution  should 
soon  disappear,  indicating  the  formation  of  soluble  starch  {amidulin) 
which  gives  a  blue  color  with  iodine.  This  body  should  soon  be  trans- 
formed into  erythrodextrin  which  gives  a  red  color  with  iodine,  and  this, 
in  turn,  should  pass  into  achroodextrin  which  gives  no  color  with  iodine. 
This  point  is  called  the  achromic  point.  When  this  point  is  reached 
test  by  Fehling's  test-  to  show  the  production  of  a  reducing  substance 
(hialtose).  A  positive  Fehling's  test  may  be  obtained  while  the 
solution  still  reacts  red  with  iodine  inasmuch  as  some  sugar  is 
formed  from  the  soluble  starch  coincidently  with  the  formation  of 
the  erythrodextrin.  For  further  discussion  of  the  transformation  of 
starch  see  p.  ~,t^. 

2.  Demonstration  of  Pancreatic  Amylase.^ — Proceed  exactly 
as  indicated  above  in  the  Demonstration  of  Salivary  Amylase  except 
that  the  saliva  is  replaced  by  5  c.c.  of  pancreatic  extract  prepared  as 
described  on  p.  141.  Pancreatic  amylase  transforms  the  starch  in  u 
manner  entirely  analogous  to  the  transformation  resulting  from  the 
action  of  sali\ary  amylase. 

3.  Preparation  of  Vegetable  Amylase. — Extract  finely  ground 
malt  with  water,  filter  and  subject  the  filtrate  to  alcoholic  fermentation 
by  means  of  yeast.  When  fermentation  is  complete  filter  off  the 
yeast  and  precipitate  the  amylase  from  the  filtrate  by  the  addition 
of  alcohol.  The  precipitate  may  be  filtered  off  and  obtained  in  the 
form  of  a  fine  white  powder. 

4.  Demonstration  of  Vegetable  Amylase. — This  enzyme  may 
be  demonstrated  according  to  the  directions  given  under  Demonstra- 
tion of  Salivary  Amylase,  p.  7,  with  the  exception  that  the  saliva 
used  in  that  experiment  is  replaced  by  an  aqueous  solution  of  the 
vegetal)le  amylase  powder  prepared  as  described  above.'* 

II.  PROTEASES. 

I.  Preparation  of  Gastric  Protease.''    Treat    the   finely   com 
minuted  mucosa  (jf  a  j^ig's  stomach  with  0.4  jjcr   cent    hydrochloric 

'  .See  p.  45. 

*  See  p.  27. 

'For  a  fliscussion  of  lliis  ciizyinc  sec  p.  j^(;. 

*  If  desired  the  first  a(|uc<)us  extract  (jf  the  oriffinal  malt  may  I"'  used  in  this  dcmoii- 
slration.     CJommen  iai  luku-diastase  may  also  lie  eiTipioycd. 

*  Also  called  pepsin,  prpsasr,  gastrii  pralriiKisr,  and  (i<  id  pnilrasr.      I'or  a  discussifm  of 
this  enzyme  see  j).  1  r  7 . 


ENZYMES   AND    THEIR  ACTION.  9 

acid  and  extract  at  38°  C.  for  24-48  hours.  The  filtrate  from  this 
mixture  constitutes  a  very  satisfactory  acid  extract  of  gastric  protease 
(see  p.  119). 

2.  Demonstration  of  Gastric  Protease. — Introduce  some  pro- 
tein material  (fibrin,  coagulated  egg-white,  or  washed  lean  beef)  into 
the  acid  extract  of  gastric  protease  prepared  as  above  described,^ 
add  an  equal  volume  of  0.4  per  cent  hydrochloric  acid  and  place  the 
mixture  at  38°  C.  for  2-3  days.  Identify  the  products  of  digestion 
according  to  directions  given  on  p.  119. 

3.  Preparation  of  Pancreatic  Protease.- — A  satisfactory  ex- 
tract of  this  enzyme  may  be  made  from  the  pancreas  of  a  pig  or  sheep 
according  to  the  directions  given  on  p.  141. 

4.  Demonstration  of  Pancreatic  Protease. — Into  an  alkaline 
extract  of  pancreatic  protease,^  prepared  as  directed  on  p.  141,  in- 
troduce some  fibrin,  coagulated  egg-white  or  lean  beef  and  place 
the  mixture  at  ;^8°  C.  for  2-5  days.^  At  the  end  of  that  period  separate 
and  identify  the  end-products  of  the  action  of  pancreatic  protease 
according  to  the  directions  given  on  p.  141. 

5.  Demonstration  of  a  Vegetable  Protease. — A  commercial 
preparation  of  papain  (papayotin,  carase  or  papase),  the  protease 
of  the  fruit  of  the  pawpaw  {carica  papaya),  may  be  used  in  this  con- 
nection. Follow  the  same  procedure  as  that  described  under  Gastric 
Protease  (see  above). 

III.  LIPASES. 

1.  Preparation  of  Pancreatic  Lipase.^ — An  extract  of  this  en- 
zyme may  be  prepared  from  the  pancreas  of  the  pig  or  sheep  accord- 
ing to  the  directions  given  on  p.  141.^ 

2.  Demonstration  of  Pancreatic  Lipase. — Into  each  of  two 
test-tubes  introduce  10  c.c.  of  milk  and  a  small  amount  of  litmus 
powder.  To  the  contents  of  one  tube  add  3  c.c.  of  a  netitral  extract 
of  pancreatic  lipase  and  to  the  contents  of  the  other  tube  add  3  c.c. 
of  a  boiled  neutral  extract  of  pancreatic  lipase.  Keep  the  tubes  at 
T,S°  C.  and  watch  for  color  changes.     The  blue  color  of  the  litmus 

'  If  so  desired,  a  solution  of  commercial  pepsin  powder  in  o .  2  per  cent  hydrochloric 
acid  may  be  substituted. 

-  Also  called  trypsin,  trypsase,  pancreatic  proteinase  and  alkali  proteinase.  For  a  dis- 
cussion of  this  enzyme  see  p.  1,58. 

■'  A  0.25  per  cent  sodium  carbonate  solution  of  commercial  trypsin  may  be  substituted. 

'  A  few  c.c.  of  toluene  or  an  alcoholic  solution  of  thymol  should  be  added  to  prevent 
putrefaction. 

■'  Also  called  steapsin.  For  a  discussion  of  this  enzyme  see  p.  130.  .\  very  active 
lipolytic  extract  may  also  be  prepared  from  the  liver. 

''  If  preferred,  a  glycerol  extract  may  be  prepared  according  to  the  directions  given  by 
Kanitz;  Zeitschrift  fiir  physiologische  Chemie,  1906,  XLVI,  p.  482. 


lO  PHYSIOLOGICAL    CHEMISTRY. 

powder  will  gradually  give  place  to  a  red.  This  change  in  the  color 
of  the  litmus  from  blue  to  red  has  been  brought  about  by  the  fatty 
acid  which  has  been  produced  through  the  lipolytic  action  exercised 
by  the  lipase  upon  the  milk  fats. 

3.  Preparation  of  Vegetable  Lipase. — This  enzyme  may  be 
readily  prepared  from  castor  beans,  several  months  old,  by  the  fol- 
lowing procedure:^  Grind  the  shelled  beans  very  fine"  and  extract 
for  twenty-four-hour  periods  with  alcohol-ether  and  ether,  in  turn. 
Reduce  the  semi-fat-free  material  to  the  finest  possible  consistency 
by  means  of  mortar  and  pestle  and  pass  this  material  through  a  sieve 
of  very  fine  mesh.  Place  this  material  in  a  Soxhlet  extractor  and 
extract  for  one  week.  This  fat-free  powder  may  then  be  used  to  dem- 
onstrate the  action  of  vegetable  lipase.  Powder  prepared  as  described 
may  be  used  in  quantitative  tests'.  For  ordinary  qualitative  tests  it 
is  not  necessary  to  remove  the  last  traces  of  fat  and  therefore  the 
extraction  period  in  the  Soxhlet  apparatus  may  be  much  shortened. 

4.  Demonstration  of  Vegetable  Lipase. — The  lipolytic  action 
of  the  lipase  prepared  from  the  castor  bean,  as  just  described,  may 
be  demonstrated  in  a  manner  entirely  analogous  to  that  used  in  the 
Demonstration  of  Pancreatic  Lipa'se,  see  p.  9.  Proceed  as  indicated 
in  that  experiment  and  substitute  the  vegetable  lipase  powder  for  the 
neutral  extract  of  pancreatic  lipase.  The  type  of  action  is  entirely 
analogous  in  the  two  instances. 

An  experiment  similar  to  Experiment  2,  p.  145,  may  also  be  tried 
if  desired.  In  this  experiment  0.2  c.c.  of  either  ethyl  butyrate  or  amyl 
acetate  may  be  employed. 

IV.  INVERTASES.' 

1.  Preparation  of  an  Extract  of  Sucrase.^ — Treat  the  finely 
divided  epithelium  of  the  small  intestine  of  a  f^.t;,  j)ig,  rat,  rabbit,  or 
hen  with  about  three  volumes  of  a  two  ])er  cent  solution  of  sodium 
lluoride  and  permit  the  mixture  to  stand  at  room  tcmjjerature  for 
twenty-four  hours.  Strain  the  extract  through  cloth  or  absorbent 
cotton  and  use  the  strained  material  in  the  following  demonstration. 

2.  Demonstration  of  Sucrase. — To  about  5  c.c.  of  a  one  per 
cent  solution  of  sucrose,  in  a  test-tube,  add  about  one  cubic  centi- 
meter of  a  two  ]jer  cent  sodium  lluoride  intestinal  extract,  prepared 

'  A.  \\.  Taylor:  On  Fcnnenlition;  L'niversity  of  Cal'Jornia  Publicaliona,  1007. 

''  The  shells  should  be  removed  witlioul  the  use  of  water.  These  heiuis  are  poisonous, 
flue  to  their  content  of  rkin. 

^  The  inverlinfi  enzymes  of  the  a/iiiirnliiry  Irart;  McikIiI  anil  Alih  lull;  A  iiuricnn  Journal 
of  Physiology,  1907-08,  XX,  p.  Si. 

*  For  a  flist  ussion  of  this  enzyme  see  p.  140. 


ENZYMES   AND    THEIR   ACTION.  II 

as  described  above.  Prepare  a  control  tube  in  which  the  intestinal 
extract  is  boiled  before  being  added  to  the  sugar  solution.  Place 
the  two  tubes  at  38°  C.  for  two  hours. ^  Heat  the  mixture  to  boiling 
to  coagulate  the  protein  material,  filter,  and  test  the  filtrate  by  Fehling's 
test  (see  p.  27).  The  tube  containing  the  boiled  extract  should  give 
no  response  to  Fehling's  test,  whereas  the  tube  containing  the  unboiled 
extract  should  reduce  the  Fehling's  solution.  This  reduction  is  due  to 
the  formation  of  invert  sugar  (see  p.  41)  from  the  sucrose  through  the 
action  of  the  enzyme  sucrase  which  is  present  in  the  intestinal  epithelium. 

3.  Preparation  of  Vegetable  Sucrase.  — Thoroughly  grind 
about  100  grams  of  brewer's  yeast  in  a  mortar  with  sand.  Spread 
the  ground  yeast  in  thin  layers  on  glass  or  porous  plates  and  dry 
it  rapidly  in  a  current  of  dry,  warm  air.  Powder  this  dry  yeast,  ex- 
tract it  with  distilled  water  and  filter.  Pour  the  filtrate  into  acetone, 
stir  and  after  permitting  the  acetone  mixture  to  stand  for  a  few  minutes 
filter  on  a  Buchner  funnel.  The  resulting  precipitate,  after  drying 
and  pulverizing,  may  be  used  to  demonstrate  vegetable  sucrase. 

4.  Demonstration  of  Vegetable  Sucrase. — To  about  5  c.c  of  a 
one  per  cent  solution  of  sucrose  in  a  test-tube  add  a  small  amount 
of  the  sucrase  powder  prepared  as  directed  above.  Place  the  tube 
at  38°  C.  for  24-72  hours  and  at  the  end  of  that  period  test  the  solution 
by  Fehling's  test.  Reduction  indicates  that  the  active  sucrase  powder 
has  transformed  the  non-reducing  sucrose  into  dextrose  and  laevulose, 
and  these  sugars,  in  turn,  have  reduced  the  Fehling  solution. 

5.  Preparation  of  an  Extract  of  Lactase.^ — Treat  the  finely 
divided  epithelium  of  the  small  intestine  of  a  kitten,  puppy,  or  pig 
embryo  with  about  three  volumes  of  a  two  per  cent  solution  of  sodium 
fluoride  and  permit  the  mixture  to  stand  at  room  temperature  for 
twenty-four  hours.  Strain  the  extract  through  cloth  or  absorbent 
cotton  and  use  the  strained  material  in  the  following  demonstration. 

6.  Demonstration  of  Lactase.^ — To  about  5  c.c.  of  a  one  per 
cent  solution  of  lactose  in  a  test-tube  add  about  one  cubic  centimeter 
of  a  toluene-water  or  a  two  per  cent  sodium  fluoride  extract  of  the 
first  part  of  the  small  intestine*  of  a  kitten,  puppy,  or  pig  embryo  pre- 
pared as  described  above.  Prepare  a  control  tube  in  which  the  intes- 
tinal extract  is  boiled  before  being  added  to  the  sugar  solution.  Place 
the  two  tubes  at  ^^^^  C.  for  24  hours.    At  the  end  of  this  period  add  one 

'  If  a  positive  result  is  not  obained  in  this  time  permit  the  digestion  to  proceed  for  a 
longer  period. 

-  For  a  discussion  of  this  enzyme  see  p.  140. 
^  Roaf;  Bio-Chemical  Journal,  igoS,  III,  p.  182. 
■'  Duodenum  and  first  part  of  jejunum. 


12  PHYSIOLOGICAL    CHEMISTRY. 

cubic  centimeter  of  the  digestion  mixture  to  5  c.c.  of  Barfoed's^  reagent 
and  place  the  tubes  in  a  boiling  water-bath.'  Examine  the  tubes  at 
the  end  of  three  minutes  against  a  black  background  in  a  good  light. 
If  no  cuprous  oxide  is  visible  replace  the  tubes  and  repeat  the  exam- 
ination at  the  end  of  the  fourth  and  fifth  minutes.  If  no  reduction  is 
then  observed  permit  the  tubes  to  stand  at  room  temperature  for  5-10 
minutes  and  examine  again. ^ 

It  has  been  determined  that  disaccharide  solutions  will  not  reduce 
Barfoed's  reagent  until  after  they  have  been  heated  for  9-10  minutes 
on  a  boiling  water-bath  in  contact  with  the  reagent.'  Therefore  in 
the  above  test,  if  the  tube  containing  the  unboiled  extract  exhibits  any 
reduction  after  being  heated  as  indicated,  for  a  period  of  five  minutes  or 
less,  and  the  control  tube  containing  boiled  extract  shows  no  reduction, 
it  may  be  concluded  that  lactase  was  present  in  the  intestinal  extract.'^ 

7.  Preparation  of  an  Extract  of  Maltase." — Treat  the  finely 
divided  epithelium  of  the  small  intestine  of  a  cat,  kitten,  or  pig  {embryo 
or  adult)  with  about  three  volumes  of  a  two  per  cent  solution  of  sodium 
fluoride  and  permit  the  mixture  to  stand  at  room  temperature  for 
twenty-four  hours.  Strain  the  extract  through  cloth  and  use  the 
strained  material  in  the  following  demonstration. 

8.  Demonstration  of  Maltase. — Proceed  exactly  as  indicated  in 
the  demonstration  of  lactase,  above,  except  that  a  one  per  cent  solu- 
tion of  maltose  is  substituted  for  the  lactose  solution.  The  extract 
used  may  be  prepared  from  the  upper  part  of  the  intestine  of  a  cat, 
kitten,  or  pig  (embryo  or  adult).  In  the  case  of  lactase,  as  indicated, 
the  intestine  used  should  be  that  of  a  kitten,  puppy,  or  pig  (embryo). 

V.  EREPSm.' 

I.  Preparation  of  Erepsin. — Grind  the  mucous  membrane  of 
the  small  intestine  of  a  cat,  dog,  or  pig  with  sand  in  a  mortar.  Treat 
the  mortared  membrane  with  toluene-  or  chloroform-water  and  permit 
the   mixture   to   stand,    with   occasional   shaking,  for   24-72    hours.** 

'  To  4.5  grams  of  neutral  crystallized  cupric  acetate  in  900  c.c.  of  water,  add  0.6  c.c. 
of  glacial  acetic  acid  and  make  the  total  volume  of  the  solution  one  liter. 

^  Care  should  be  taken  to  see  that  the  water  in  the  hath  reaches  at  least  to  the  ujjpcr 
level  of  the  c<jntents  oi  the  tubes. 

*  Sometimes  the  drawinj^  of  conclusions  is  facililalcd  Ijy  iKiurin^^  the  niixtun-  from  the 
lube  and  examining  the  bottom  of  the  tube  for  adherent  cujjrous  oxide. 

*  The  heating  for  9-10  minutes  is  sufficient  t<j  transform  the  di,sa(  ( haridt-  int(j  mono- 
saccharide. 

'  The  reduc  li(in  would,  of  course,  be  due  to  the  action  of  the  dextrose  and  galactose 
which  had  been  formed  fnjm  the  lactose  through  the  action  of  the  enzyme  lactase. 

"  For  a  discussion  of  this  enzyme  see  p.  54. 

'  .'\lsfi  called  erep.sase.     I'or  a  rlistussion  of  this  enzyme  sc-e  p.  140. 

''  The  enzyme  may  also  be  extra(  ted  by  means  of  glycmil  01  nlkdlhic  " /ihysiolof^ical" 
suit  solution  if  desircfl. 


ENZYMES   AND    THEIR  ACTION.  1 3 

Filter  the  extract  thus  prepared  through  cotton  and  use  the  filtrate  in 
the  following  experiment. 

2.  Demonstration  of  Erepsin. — To  about  5  c.c.  of  a  one  per 
cent  solution  of  Witte's  peptone  in  a  test-tube  add  about  one  c.c.  of 
the  erepsin  extract  prepared  as  described  above  and  make  the  mixture 
slightly  alkaline  (o.i  per  cent)  with  sodium  carbonate.  Prepare  a 
second  tube  containing  a  like  amount  of  peptone  solution  but  hoil 
the  erepsin  extract  before  introducing  it.  Place  the  two  tubes  at  2)^° 
C.  for  2-3  days.  At  the  end  of  that  period  heat  the  contents  of  each 
tube  to  boiling,  filter  and  try  the  biuret  test  on  each  filtrate.  In  making 
these  tests  care  should  be  taken  to  use  like  amounts  of  filtrate,  potas- 
sium hydroxide  and  cupric  sulphate  in  each  test  in  order  that  the 
drawing  of  correct  conclusions  may  be  facihtated.  The  contents  of 
the  tube  which  contained  the  boiled  extract  should  show  a  deep  pink 
color  with  the  biuret  test,  due  to  the  peptone  still  present.  On  the 
other  hand,  the  biuret  test  upon  the  contents  of  the  tube  containing 
the  unboiled  extract  should  be  negative  or  exhibit,  at  the  most,  2^  faint 
pink  or  blue  color,  signifying  that  the  peptone,  through  the  influence 
of  the  erepsin,  has  been  transformed,  in  great  part  at  least,  into  amino 
acids  which  do  not  respond  to  the  biuret  test.  ^ 

VI.  URICOLYTIC  ENZYME.= 

1.  Preparation  of  Uricolytic  Enzyme. — Extract  pulped  liver 
tissue  with  toluene-  or  chloroform-water  at  38°  C.  for  24  hours,  with 
occasional  shaking.  Filter  the  extract  and  use  the  filtrate  in  the 
following  experiment. 

2.  Demonstration  of  Uricolytic  Enzyme. — Add  about  o.i 
gram  of  uric  acid  to  10  c.c.  of  water  and  bring  the  uric  acid  into  solu- 
tion by  the  addition  of  the  minimal  quantity  of  potassium  hydroxide. 
To  5  c.c.  of  this  uric  acid  solution,  in  a  test-tube,  add  5  c.c.  of  the 
uricolytic  enzyme  extract  prepared  as  described  above.  Prepare  a 
second  tube  containing  a  like  amount  of  uric  acid  solution,  but  boil 
the  extract  before  it  is  introduced.  Place  the  two  tubes  at  38°  C.  for 
3-4  days  and  titrate  the  two  digestive  mixtures  with  a  solution  of 
potassium  permanganate  according  to  directions  given  under  Folin- 
Shaffer  Method,  Chapter  XXII.  It  will  be  found  that  the  mixture 
containing  the  boiled  extract  requires  a  much  larger  volume  of  the 

'  Strictly  speaking,  this  erepsin  demonstration  is  not  adequate  unless  a  control  test  is 
made  with  native  protein  (except  caseinogen,  histones  and  protamines)  to  show  that  the 
extract  is  trypsin-free  and  digests  peptone  but  not  native  protein. 

-  Mendel  and  Mitchell;  American  Journal  of  Physiology,  1908,  XX,  p.  07. 


14  PHYSIOLOGICAL    CHEMISTRY. 

permanganate  to  complete  the  titration  than  the  other  tube.  This 
indicates  that  a  uricolytic  enzyme  has  destroyed  at  least  a  portion  of  the 
uric  acid  which  was  originally  present  in  the  tube  containing  the 
imhoiled  extract. 

VII.  CATALASE. 
Demonstration  of  Catalase. — The  various  animal  tissues,  such 
as  liver,  kidney,  blood,  lung,  muscle  and  brain,  contain  an  enzyme 
called  catalase  which  possesses  the  property  of  decomposing  hydro- 
gen peroxide.  The  presence  of  this  enzyme  may  be  demonstrated 
as  follows:  Introduce  into  a  low,  broad,  wide-mouthed  bottle  some 
pulped  liver  tissue  and  a  porcelain  crucible  containing  neutral  hydro- 
gen peroxide.^  Connect  the  bottle  with  a  eudiometer  filled  with 
water,  upset  the  crucible  of  hydrogen  peroxide  upon  the  liver  pulp 
and  note  the  collection  of  gas  in  the  eudiometer.  This  gas  is  oxygen 
which  has  been  liberated  from  the  hydrogen  peroxide  through  the 
action  of  the  catalase  of  the  liver  tissue. 

B.    Experiments  on  Anti-Enz3mies. 

1.  Preparation  of  an  Extract  of  Anti-Pepsin.^ — Grind  up  a 
number  of  intestinal  worms  (ascaris)^  with  quartz  sand  in  a  mortar. 
Subject  this  mass  to  high  pressure,  filter  the  resultant  juice  and  treat 
it  with  alcohol  until  a  concentration  of  sixty  per  cent  is  reiached. 
If  any  precipitate  forms  it  should  be  filtered  off*  and  alcohol  added 
to  the  filtrate  until  the  concentration  of  alcohol  is  85  per  cent,  or  over. 
The  anti-enzyme  is  precipitated  by  this  concentration.  Permit  this 
precipitate  to  stand  for  twenty-four  hours,  then  filter  it  off,  wash  it 
with  95  per  cent  alcohol,  absolute  alcohol,  and  ether,  in  turn,  and 
finally  dry  the  substance  over  sulphuric  acid.  The  sticky  powder 
which  results  may  be  used  in  this  form  or  may  be  dissolved  in  water 
as  desired  and  the  aqueous  solution  used/' 

2.  Demonstration  of  Anti-Pepsin." — Introduce  into  a  test-tube 
a  few  fibrin  shreds  and  c([ual  volumes  of  pepsin-hydrochloric  acid^ 
and  ascaris  extract  made  as  indicated  above.  Prepare  a  control  tube 
in    which    the    ascaris    extract    is    replaced    by    water.     Place    the 

'  Mendel  and  Leavenworth;  American  Jojirnal  of  Physiology,  igo8,  XXI,  p.  85. 

^  Anti-gastric-proteasc  or  anti-af.id-proleasc. 

^  'J'hesc  may  be  readily  ohtained  from  pij^s  at  a  slaughter  house. 

*  This  precipitate  consists  of  impurities,  tlie  anli-cnzyme  not  being  precipitated  until  a 
higher  concentration  of  alcohol  is  reached. 

'  The  original  ascaris  extract  possesses  much  greater  activit}'  than  cither  the  powder 
or  the  af|ueous  solution. 

"  Martin  11.  Fischer;  Physiology  of  Alimentation,  1007,  p.  134. 

'  Made  by  bringing  0.015  gram  of  pepsin  into  solution  in  7  c.c.  of  water  and  0.23  gram 
of  concentrated  hydrochloric  acid. 


ENZYMES  AND   THEIR  ACTION.  1 5 

tubes  at  38°  C.  Ordinarily  in  one  hour  the  fibrin  in  the  control  tube 
will  be  completely  digested.  The  fibrin  in  the  tube  containing  the 
ascaris  extract  may,  however,  remain  unchanged  for  days,  thus 
indicating  the  inhibitory  influence  exerted  by  the  anti-enzyme  pres- 
ent in  this  extract. 

3.  Preparation  of  an  Extract  of  Anti-Trypsin/ — The  extract 
may  be  prepared  from  the  intestinal  worm,  ascaris,  according  to  the 
directions  given  on  page  14. 

4.  Demonstration  of  Anti-Trypsin.— Introduce  into  a  test-tube 
a  few  shreds  of  fibrin  and  equal  volumes  of  an  artificial  tryptic  solu- 
tion^ and  the  ascaris  extract  made  as  described  on  page  14.  Prepare 
a  control  tube  in  which  the  ascaris  extract  is  replaced  by  water.  Place 
the  two  tubes  at  ^,8°  C.  Ordinarily  the  fibrin  in  the  control  tube  will 
be  completely  digested  in  two  hours.  The  fibrin  in  the  tube  contain- 
ing the  ascaris  extract  may,  however,  remain  unchanged  for  days,  thus 
indicatihg  the  inhibitory  influence  of  the  anti-enzyme. 

Blood  serum  also  contains  anti-try psin.  This  may  be  demon- 
strated as  follows:  Introduce  equal  volumes  of  serum  and  artificial 
tryptic  solution  (prepared  as  described  above)  into  a  test-tube  and 
add  a  few  shreds  of  fibrin.  Prepare  a  control  tube  containing  boiled 
serum.  Place  the  two  tubes  at  ^S°  C.  It  will  be  observed  that  the 
fibrin  in  the  tube  containing  the  boiled  serum  digests,  whereas  that 
in  the  other  tube  does  not  digest.  The  anti-trypsin  present  in  the 
unboiled  serum  has  exerted  an  inhibitory  influence  upon  the  action 
of  the  trypsin. 

C.    Quantitative  Applications. 

I.  Quantitative  Determination  of  Amylolytic  Activity. — Wohl- 
gemuth's  Method. — Arrange  a  series  of  test-tubes  with  diminishing 
quantities  of  the  enzyme  solution  under  examination,  introduce  into 
each  tube  5  c.c.  of  a  i  per  cent  solution  of  soluble  starch^  and  place 
each  tube  at  once  in  a  bath  of  ice-water.*    When  all  the  tubes  have 

'  Anti-pancreatic-protease  or  anti-alkali-protease. 

-Made  by  dissolving  0.04  gram  of  sodium  carbonate  and  0.015  gram  of  trypsin  in 
S  c.c.  of  water. 

'  Kahlbaum's  soluble  starch  is  satisfactory.  In  preparing  the  1  per  cent  solution,  the 
weighed  starch  powder  should  be  dissolved  in  the  proper  volume  of  cold  distilled  water 
and  stirred  until  a  homogeneous  suspension  is  obtained.  The  mixture  should  then  be 
heated,  with  constant  stirring,  in  a  porcelain  dish,  until  it  is  clear.  This  ordinarily  takes 
about  8-10  minutes.  A  slightly  opaque  solution  is  thus  obtained  which  should  be  cooled 
before  using. 

*  Ordinarily  a- series  of  six  tubes  is  satisfactory,  the  volumes  of  the  enzyme  solution  used 
ranging  from  i  c.c.  to  o.i  c.c.  and  the  measurements  being  made  by  means  of  a  i  c.c. 
graduated  pipette.  Each  tube  should  be  paced  in  the  ice-water  bath  as  soon  as  the  starch 
solution  is  introduced.  It  will  be  found  convenient  to  use  a  small  wire  basket  to  hold  the 
tubes. 


1 6  PHYSIOLOGICAL    CHEMISTRY. 

been  prepared  in  this  way  and  placed  in  the  ice- water  bath  they  are 
transferred  to  a  water-bath  or  incubator  and  kept  at  38°  C.  for  from 
thirty  minutes  to  an  hour/  At  the  end  of  this  digestion  period  the 
tubes  are  again  removed  to  the  bath  of  ice  water  in  order  that  the 
action  of  the  enzyme  may  be  stopped. 

Dilute  the  contents  of  each  tube,  to  within  about  one-half  inch 
of  the  top,  with  water,  add  one  drop  of  a  N/io  solution  of  iodine  and 
shake  the  tube  and  contents  thoroughly.  A  series  of  colors  ranging 
from  dark  blue  through  bluish-violet  and  reddish-yellow  to  yellow,  will 
be  formed."  The  dark  blue  color  shows  the  presence  of  unchanged 
starch,  the  bluish-violet  indicates  a  mixture  of  starch  and  erythro- 
dextrin,  whereas  the  reddish-yellow  signifies  that  erythrodextrin  and 
maltose  are  present,  and  the  yellow  solution  denotes  the  complete 
transformation  of  starch  into  maltose.  Examine  the  tubes  carefully 
before  a  white  background  and  select  the  last  tube  in  the  series  which 
shows  the  entire  absence  of  all  blue  color,  thus  indicating  that  the  starch 
has  been  completely  transformed  into  dextrins  and  sugar.  In  case 
of  indecision  between  two  tubes,  add  an  extra  drop  of  the  iodine 
solution,  and  observe  them  again,  after  shaking. 

Calculatimi. — The  amylolytic  activity^  of  a  given  solution  is  ex- 
pressed in  terms  of  the  activity  of  i  c.c.  of  such  a  solution.  For  ex- 
ample, if  it  is  found  that  0.02  c.c.  of  an  amylolytic  solution,  acting 
at  38°  C,  completely  transformed  the  starch  in  5  c.c.  of  a  i  per  cent 
starch  solution  in  30  minutes,  the  amylolytic  activity  of  such  a  solution 
would  be  expressed  as  follows: 

Z>38°=-.2C50. 

30        J 

This  indicates  that  i  c.c.  of  the  solution  under  examination  possesses 
the  power  of  completely  digesting  250  c.c.  of  a  r  per  cent  starch  solu- 
tion in  30  minutes  at  38^^  C. 

2.  Quantitative  Determination  of  Peptic  Activity,  (a)  Metl's 
Method. — The  determination  of  the  actual  rate  of  peptic  activity  is 
a  most  important  procedure  under  certain  conditions.  Several  meth- 
ods of  making  this  determination  are  in  use.  The  method  of  Sprigg'* 
is  probably  the  most  accurate  method  yet  devised  for  this  purpose. 
It    is,    however,    too    complicated    and    time  consuming    for    clinical 

'  Longer  digestion  periods  may  he  used  where  it  is  deemed  advisal)Ie.  If  exceedingly 
weak  sfjiutiops  are  being  investigatefl,  it  may  he  most  satisfactory  to  permit  the  digestion 
to  extcpfl  over  a  periori  of  24  hours. 

^Sce  p.  4S. 

^  Designated  by  "  I)"  the  first  letter  of  "diastatic." 

*  Sprigg:  Zeitschrift  fiir  physiolof^isrlte  C'hcmie,  i(j02,  XX.W,  |j.  465. 


ENZYMES   AND    THEIR   ACTION.  1 7 

purposes.  The  method  of  Mett,  given  below,  is  very  simple  although 
not  strictly  accurate.  The  procedure  is  as  follows:  To  about  5  c.c. 
of  the  gastric  juice  under  examination  in  a  test-tube  add  a  section  of 
a  Mett  tube ^  and  place  the  mixture  at  ^8°  C.  for  ten  hours.  At  the  end 
of  this  period,  the  tube  should  be  removed  from  the  gastric  juice  and 
the  length  of  the  column  of  coagulated  albumin  which  has  been 
digested  carefully  determined  by  means  of  a  low-power  microscope  and 
a  millimeter  scale.  In  normal  human  gastric  juice  the  upper  limit  is 
4  mm.  However,  control  tests  should  always  be  made  to  determine 
the  digestibility  of  the  coagulated  albumin  in  artificial  gastric  juice, 
inasmuch  as  this  factor  will  vary  with  different  albumin  preparations. 

In  connection  with  this  test  Schiitz's  law  should  be  borne  in  mind. 
This  principle  is  to  the  effect  that  the  amount  of  proteolytic  enzyme 
present  in  a  digestion  mixture  is  proportional  to  the  square  of  the  number 
of  millimeters  of  albumin  digested.  Therefore  a  gastric  juice  which 
digests  2  mm.  of  albumin  contains  four  times  as  much  pepsin  as  a 
gastric  juice  which  digests  only  i  mm.  of  albumin.  And  further,  if 
the  quantities  of  albumin  digested  are  2  mm.  and  3  mm.,  respectively, 
the  ratio  between  the  pepsin  values  will  be  as  4:9. 

It  is  claimed  by  Nirenstein  and  Schiff  that  the  principle  of  Schiitz 
does  not  apply  to  gastric  juice  unless  this  fluid  be  diluted  with  fifteen 
volumes  of  N/20  hydrochloric  acid. 

{h)  Fuld  and  Levison's  Method. — This  test  is  founded  upon  the 
fact,  shown  by  Osborne,  that  edestin  when  brought  into  solution  in 
dilute  acid  will  change  in  its  solubility,  due  to  the  contact  with  the 
acid,  and  that  a  protean  called  edestan,  which  is  insoluble  in  neutral 
fluid,  will  be  formed.  The  procedure  is  as  follows:  Dilute  the  gastric 
juice  under  examination  with  20  volumes  of  water  and  introduce 
gradually  decreasing  volumes  of  the  diluted  juice  into  a  series^  of 
narrow  test-tubes  about  i  cm.  in  diameter.  The  measurements  of 
gastric  juice  may  conveniently  be  made  with  a  one  c.c.  pipette  which 
is  accurately  graduated  in  i/ioo  c.c.  Into  the  first  tube  in  the  series 
may  be  introduced  one  c.c.   of  gastric  juice,  and  the  tubes  which 

'  In  the  preparation  of  these  tubes,  egg-white  is  diluted  with  an  equal  volume  of  water, 
the  precipitated  globulin  filtered  off  and  the  filtrate  collected  in  a  tall,  narrow  beaker  or  a 
large  test-tube.  A  bundle  of  capillary  tubes  about  lo  cm.  in  length  and  2  mm.  in  diameter 
are  now  placed  in  this  vessel  in  such  a  manner  that  they  are  completely  submerged  in  the 
albumin  solution.  After  an  examination  has  shown  that  the  tubes  are  completely  filled 
with  the  albumin  solution  and  that  there  are  no  interfering  air-bubbles,  the  vessel  and  its 
contained  tubes  is  heated  for  5-15  minutes  in  a  boiling  water-bath,  in  order  to  coagulate 
the  albumin.  When  this  coagulation  is  complete,  the  tubes  are  removed,  all  albumin 
adhering  to  them  is  carefully  cleaned  off,  and  the  tubes  rendered  air-tight  by  the  application 
of  sealing  wax  at  either  end.  When  needed  for  use,  these  tubes  are  cut  into  sections  about 
2  cm.  in  length. 

-  The  longer  the  series,   the  more  accurate  the  deductions  which  may  be  drawn. 


1 8  PHYSIOLOGICAL    CHEMISTRY. 

follow  in  the  series  may  receive  volumes  which  differ,  in  each  instance, 
from  the  volume  introduced  into  the  preceding  tube  by  i/ioo,  1/50, 
1/20,  or  i/io  of  a  cubic  centimeter.  Now  rapidly  introduce  into 
each  tube  the  same  volume  {e.  g.,  2  c.c.)  of  a  i :  1000 solution  of  edestin^ 
and  place  the  tubes  at  40°  C.  for  one-half  hour.  At  the  end  of  this 
time  stratify  ammonium  hydroxide  upon  the  contents  of  each  tube," 
place  the  tubes  in  position  before  a  black  background  and  examine 
them  carefully.  The  ammonium  hydroxide,  by  diffusing  into  the  acid 
fluid,  forms  a  neutral  zone  and  in  this  zone  will  be  precipitated  any 
undigested  edestan  which  is  present.  Select  the  tube  in  the  series 
which  contains  the  least  amount  of  gastric  juice  and  which  exhibits 
no  ring,  signifying  that  the  edestan  has  been  completely  digested,  and 
calculate  the  peptic  activity  of  the  gastric  juice  under  examination 
on  the  basis  of  the  volume  of  gastri'c  juice  used  in  this  particular  tube. 

Calculation. — Multiply  the  number  of  c.c.  of  edestin  solution 
used  by  the  dilution  to  which  the  gastric  juice  was  originally  subjected 
and  divide  the  volume  of  gastric  juice  necessary  to  completely  digest 
the  edestan  by  this  product.  For  example,  if  2  c.c.  of  the  edestin 
solution  was  completely  digested  by  0.25  c.c.  of  a  1:20  gastric  juice 
we  would  have  the  following  expression:  0:25  ^20X2  or  1:160.  This 
peptic  activity  may  be  expressed  in  several  ways,  e.  g.,  (a)  i :  160  pepsin ; 
(h)  160  pepsin  content;  (c)  160  parts. 

3.  Quantitative  Determination  of  Tryptic  Activity. — Gross' 
Method. — -This  method  is  based  upon  the  principle  that  faintly  alka- 
line solutions  of  casein  are  precipitated  upon  the  addition  of  dilute 
(i  per  cent)  acetic  acid  whereas  its  digestion  products  are  not  so 
precipitated.  The  method  follows:  Prepare  a  series  of  tubes  each 
containing  10  c.c.  of  a  o.i  per  cent  solution  of  pure,  fat-free  casein,'' 
which  has  been  heated  to  a  temperature  of  40°  C.  Add  to  the  con- 
tents of  the  series  of  tubes  increasing  amounts  of  the  trypsin  solution 
under  examination,''  and  place  them  at  40°  C.  for  fifteen  mimilcs. 
At  the  end  of  this  time  remove  the  tubes  and  acidify  the  contents  of 
each  with  a  few  drops  of  dilute  (i  per  cent)  acetic  acid.     The  tubes 

'  This  edcslin  should  \n-  |)R-|j;irc(l  in  llic  usiuil  way  (sec  p.  loi),  unci  lnoiif^lit  into 
solution  in  a  dilute  liydnx  liloric  acid  of  aiiproximalely  the  same  slren^^lh  as  that  whicli 
occurs  normally  in  ihe  luiman  stomach.  This  may  lie  conveniently  made  liy  a(]<ling  .^o  c.c. 
of  N/io  hydro!  lilorif  add  to  70  c.c.  of  water.  Ordinarily  it  should  not  tal-.e  longer  than 
one  minute  to  introduce  the  edestin  solution  into  the  entire  series  of  tubes.  However,  if 
the  ofiestin  is  addefl  to  the  tuhes  in  the  same  order  as  the  ammonium  hydro,\ide  is  afterward 
stratified,  no  apprec:ial)le  error  is  introduced. 

'''  Making  the  stratification  in  the  same  order  as  the  edestin  solution  was  added. 

''Made  by  <li.ssolving  one  gram  of  driihler's  casein  in  a  liter  of  o.i  per  i cnt  sotlium 
carbonate.     ,'\  little  r  hloroform  may  be  added  to  prevent  bacterial  action. 

•The  amount  of  solution  used  nuiy  vary  from  o.t-i  i-x.  The  measiirenicnis  may 
conveniently  be  mafic  by  means  f)f  a  i  c.c.  graduated  pipette. 


ENZYMES   AND    THEIR  ACTION.  1 9 

in  which  the  casein  is  completely  digested  will  remain  clear  when 
acidified,  while  those  tubes  which  contain  undigested  casein  will  become 
more  or  less  turbid  under  these  conditions.  Select  the  first  tube  in  the 
series  which  exhibits  no  turbidity  upon  acidification,  thus  indicating 
complete  digestion  of  the  casein,  and  calculate  the  tryptic  activity  of 
the  enzyme  solution  under  examination. 

Calculation. — The  unit  of  tryptic  activity  is  an  expression  of  the 
power  of  I  c.c.  of  the  fluid  under  examination  exerted  for  a  period 
of  fifteen  minutes  on  lo  c.c.  of  a  o.i  per  cent  casein  solution.  For 
example,  if  0.5  c.c.  of  a  trypsin  solution  completely  digests  10  c.c. 
of  a  0.1  per  cent  solution  of  casein  in  fifteen  minutes  the  activity  of 
that  solution  would  be  expressed  as  follows: 

Tryptic  activity  =  i -^o. 5  =  2. 

Such  a  trypsin  solution  would  be  said  to  possess  an  activity  of 
2.  If  0.3  c.c.  of  the  trypsin  solution  had  been  required  the  solution 
would  be  said  to  possess  an  activity  of  3.3;  i.  e.,  1-^0.3  =  3.3. 


CHAPTER   II. 

CARBOHYDRATES. 

The  name  carbohydrates  is  given  to  a  class  of  bodies  which  arc 
an  especially  prominent  constituent  of  plants  and  which  are  found 
also  in  the  animal  body  either  free  or  as  an  integral  part  of  various 
proteins.  They  are  called  carbohydrates  because  they  contain  the 
elements  C,  H  and  O;  the  H  and  O  being  present  in  the  proportion 
to  form  water.  The  term  is  not  strictly  appropriate  inasmuch  as 
there  are  bodies,  such  as  acetic  acid,  lactic  acid  and  inosite,  which 
have  H  and  O  present  in  the  proportion  to  form  water,  but  which 
are  not  carbohydrates,  and  there  are  also  true  carbohydrates  which 
do   not   have  H  and  O  present  in  this  proportion,  e.  g.,  rhamnose, 

Chemically  considered,  the  carbohydrates  are  aldehyde  or  ketone 
derivatives  of  complex  alcohols.  Treated  from  this  standpoint,  the 
aldehyde  derivatives  are  spoken  of  as  aldoses,  and  the  ketone  deriva- 
tives are  spoken  of  as  ketoses.  The  carbohydrates  are  also  frequently 
named  according  to  the  number  of  oxygen  atoms  present  in  the  mole- 
cule, e.  g.,  trioscs,  pentoses,  and  hexoses. 

The  more  common  carbohydrates   may  be  classified   as   follows: 

I.  Monosaccharides. 

1.  Hexoses,  CgHj20g. 

(a)  Dextrose. 

(b)  Laevulose. 

(c)  Galactose. 

2.  Pentoses,  CsH^Oj. 

(a)  Arabinose. 

(b)  Xylose. 

(c)  Rhamnose  (Methyl-jjcntose),  C.JIi./X,. 

II.  hisaccharides,  C ^^U.^^O ^^. 

1.  Maltose. 

2.  Lactose. 

3.  Iso-Maltose. 

4.  Sucrose. 

20 


CARBOHYDRATES.  21 


III.  Trisaccharides,   Cj8H320jg. 

I. 

Raffinose. 

IV.  Polysaccharides,  (C.H^.O^)^. 

I. 

Starch  Group. 

(a)  Starch. 

{b)  Inulin. 

(c)  Glycogen. 

(d)  Lichenin. 

2. 

Gum  and  Vegetable  Mucilage  Group 

(a)  Dextrin. 

(b)  Vegetable  Gums. 

3- 

Cellulose  Group. 

(a)  Cellulose. 

(b)  Hemi-Cellulose. 

Each  member  of  the  above  carbohydrate  classes,  except  the  mem- 
bers of  the  pentose  group,  may  be  supposed  to  contain  the  group 
CgHjgOg,  called  the  saccharide  group.  The  polysaccharides  consist 
of  this  group  alone  taken  a  large  number  of  times,  whereas  the  disac- 
charides  may  be  supposed  to  contain  two  such  groups  plus  a  molecule 
of  water,  and  the  monosaccharides  to  contain  one  such  group  plus 
a  molecule  of  water.  Thus,  (CgHjo05)3^  =  polysaccharide,  (CeHjo05)2 
+  H20  =  disaccharide,  CgHj^Og  +  HgO^  monosaccharide.  In  a 
general  way  the  solubility  of  the  carbohydrates  varies  with  the  number 
of  saccharide  groups  present,  the  substances  containing  the  largest 
number  of  these  groups  being  the  least  soluble.  This  means  simply 
that,  as  a  class,  the  monosaccharides  (hexoses)  are  the  most  soluble 
and  the  polysaccharides  (starches  and  cellulose)  are  the  least  soluble. 


MONOSACCHARIDES. 

Hexoses,  CgHj20g. 

The  hexoses  are  monosaccharides  containing  six  oxygen  atoms 
in  a  molecule.  They  are  the  most  important  of  the  simple  sugars, 
and  two  of  the  principal  hexoses,  dextrose  and  laevulose,  occur  widely 
distributed  in  plants  and  fruits.  Of  these  two  hexoses,  dextrose 
results  from  the  hydrolysis  of  starch  whereas  both  dextrose  and 
laevulose  are  formed  in  the  hydrolysis  of  sucrose.  Galactose,  which 
with  dextrose  results  from  the  hydrolysis  of  lactose,  is  also  an  impor- 
tant hexose.     These  three  hexoses  are  fermentable  by  yeast,  and  yield 


22  PHYSIOLOGICAL    CHEMISTRY. 

laevulinic  acid  upon  heating  with  dikite  mineral  acids.  They  reduce 
metalHc  oxides  in  alkahne  sokition,  are  optically  active  and  extremely 
soluble.     With  phenylhydrazine  they  form  characteristic  osazones. 

CH,OH 

I 
DEXTROSE,  (CHOH),. 

CHO 

Dextrose,  also  called  glucose  or  grape  sugar,  is  present  in  the  blood 
in  small  amount  and  may  also  occur  in  traces  in  normal  urine.  After 
the  ingestion  of  large  amounts  of  sucrose,  lactose  or  dextrose,  causing 
the  assimilation  limit  to  be  exceeded,  an  alimentary  glycosuria  may 
arise.  In  diabetes  mellitus  very  large  amounts  of  dextrose  are 
excreted  in  the  urine.  The  following  structural  formula  has  been 
suggested  by  Victor  Meyer  for  ^/-dextrose: 

COH 
H-C-OH 

HO-C-H 

I 
H-C-OH 

H-C-OH 

CH^OH 

(For  further  discussion  of  dextrose  see  section  on  Hexoses,  page  21.) 

Experiments  on  Dextrose. 

i.   Solubility.     Test  the  solubility  of  dextrose  in  the  "ordinary 
solvents"  and  in  alcohol.     (In  the  solubility  tests  throughout  the  book 
we  shall  designate  the  following  solvents  as  the  "ordinary  solvents": 
H.^O;  10  per  cent  NaCl;  0.5  per  cent  NajCO,;  0.2  per  cent  HCl;  con 
centrated  KOH;  concentrated  HCl.) 

2.  Molisch's  Reaction.  Place  approximately  5  c.c.  of  concen- 
trated H^SO^  in  a  test-tube.  Incline  the  tube  and  slowly  pour  down 
the  inner  side  of  it  apj^roximately  5  c.c.  of  the  sugar  solution  to  which 
2  flro[>s  of  Molisch's  reagent  (a  i  5  y)er  cent  alcoholic  solution  of  a-naph 


PLATE  III. 


()SAZ(JNS. 

U\j\Htr  form,    dcxlrosa/.on;  ccnir.il   form,   m;illos;i/,oii;   low(;r  fonii,    hu  losazoii. 


CARBOHYDRATES.  23 

thol)  has  been  added,  so  that  the  sugar  solution  will  not  mix  with  the 
acid.  A  reddish- violet  zone  is  produced  at  the  point  of  contact.  The 
reaction  is  due  to  the  formation  of  furfurol, 

HC-CH 

HC     CCHO, 

O 

by  the  acid.  The  test  is  given  by  all  bodies  containing  a  carbohydrate 
group  and  is  therefore  not  specific  and,  in  consequence,  of  very 
little  practical  importance. 

3.  Phenylhydrazine  Reaction. — Test  according  to  one  of  the 
following  methods:  (a)  To  a  small  amount  of  phenylhydrazine 
mixture,  furnished  by  the  instructor,^  add  5  c.c.  of  the  sugar  solution, 
shake  well  and  heat  on  a  boiling  water-bath  for  one-half  to  three-quar- 
ters of  an  hour.  Allow  the  tube  to  cool  slowly  and  examine  the  crystals 
microscopically  (Plate  III,  opposite).  If  the  solution  has  become  too 
concentrated  in  the  boiling  process  it  will  be  light  red  in  color  and  no 
crystals  will  separate  until  it  is  diluted  with  water. 

Yellow  crystalline  bodies  called  osazones  are  formed  from  certain 
sugars  under  these  conditions,  in  general  each  individual  sugar  giving 
rise  to  an  osazone  of  a  definite  crystalline  form  which  is  typical  for 
that  sugar.  It  is  important  to  remember  in  this  connection  that  of 
the  simple  sugars  of  interest  in  physiological  chemistry,  dextrose  and 
laevulose  yield  the  same  osazone.  Each  osazone  has  a  definite  melting- 
point  and  as  a  further  and  more  accurate  means  of  identification  it 
may  be  recrystallized  and  identified  by  the  determination  of  its  melting- 
point  and  nitrogen  content.  The  reaction  taking  place  in  the  forma- 
tion of  phenyldextr osazone  is  as  follows : 

C,H,30,  +  2(H3N-NH-C,H,)  =  C3H,,0,(N-NH-CeH3),-f2H30  +  H3. 

Dextrose.  Phenylhydrazine.  Phenyldextrosazone. 

(b)  Place  5  c.c.  of  the  sugar  solution  in  a  test-tube,  add  i  c.c  of  the 
phenylhydrazine-acetate  solution  furnished  by  the  instructor,^  and 
heat  on  a  boiling  water-bath  for  one-half  to  three-quarters  of  an  hour. 
Allow  the  liquid  to  cool  slowly  and  examine  the  crystals  microscopically 
(Plate  III,  opposite). 

^  This  mixture  is  prepared  by  combining  one  part  of  phenylhydrazine  hydrochloride 
and  two  parts  of  sodium  acetate,  by  weight.     These  are  thoroughly  mixed  in  a  mortar. 

^  This  solution  is  prepared  by  mixing  one  part  by  volume,  in  each  case,  of  glacial  acetic 
acid,  one  part  of  water  and  two  parts  of  phenylhydrazine  (the  base). 


24  PHYSIOLOGICAL    CHEMISTRY. 

The  phenylhydrazine  test  has  been  so  modified  b}'  Cipollina  as  to 
be  of  use  as  a  rapid  clinical  lest.  The  directions  for  this  test  are  given 
in  the  next  experiment. 

4.  Cipollina's  Test. — Thoroughly  mix  4  c.c.  of  dextrose  soki- 
tion,  5  drops  of  phenylhydrazine  (the  base)  and  1/2  c.c.  of  glacial 
acetic  acid  in  a  test-tube.  Heat  the  mixture  for  about  one  minute  over 
a  low  fiame,  shaking  the  tube  continually  to  prevent  loss  of  fluid  by 
bumping.  Add  4-5  drops  of  sodium  hydroxide  (sp.  gr.  1.16),  being 
certain  that  the  fluid  in  the  test-tube  remains  acid,  heat  the  mixture 
again  for  a  moment  and  then  cool  the  contents  of  the  tube.  Ordinarily 
the  crystals  form  at  once,  especially  if  the  sugar  solution  possesses  a 
low  specific  gravity.  If  they  do  not  appear  immediately  allow  the 
tube  to  stand  at  least  20  minutes  before  deciding  upon  the  absence  of 
siigar. 

Examine  the  crystals  under  the  microscope  and  compare  them 
with  those  shown  in  Plate  III,  opposite  page  23. 

5.  Riegler's  Reaction.^ — Introduce  o.i  gram  of  phenylhydra- 
zine-hydrochloride  and  0.25  gram  of  sodium  acetate  into  a  test-tube, 
add  20  drops  of  the  solution  under  examination  and  heat  the  mixture 
to  boiling.  Now  introduce  10  c.c.  of  a  3  per  cent  solution  of  potassium 
hydroxide  and  gently  shake  the  tube  and  contents.  If  the  solution 
under  examination  contains  dextrose  the  liquid  in  the  tube  will  assume 
a  red  color.  One  per  cent  dextrose  yields  an  immediate  color  whereas 
0.05  per  cent  yields  the  color  only  after  the  lapse  of  a  period  of  one- 
half  hour  from  the  time  the  alkali  is  added.  In  urinary  examination 
if  the  color  appears  after  the  thirty-minute  interval  the  color  change  is 
without  significance  inasmuch  as  sugar-free  urine  will  respond  thus. 
The  reaction  is  given  by  all  aldehydes  and  therefore  the  test  cannot 
be  safely  employed  in  testing  urines  preserved  by  formaldehyde. 
Albumin  does  not  interfere  with  the  test. 

,  6.  Bottu's  Test.-— To  8  c.c.  of  Bottu's  reagent^*  in  a  test-tube 
add  I  c.c.  of  the  solution  under  examination  and  mix  the  li([uids  by 
gentle  shaking.  Now  heat  the  upper  portion  of  the  mixture  to  boiling, 
add  an  additional  1  c.c.  of  the  solution  and  heat  the  mixture  again 
immediately.  The  appearance  of  a  blue  color  accompanied  by  the 
precipitation  of  small  particles  of  indigo  blue  indicates  the  presence  of 
flextro.se  in  the  solution  under  examination.  The  test  will  serve  to 
detect  the  presence  of  o.  r  per  cent  of  dextrose. 

•    '  Kieglcr;  Compl.  rciul.  s<k:.  biol.,  66,  p.  705. 

^Bottu;  Com|jl.  ri-iifl.  s<k  .  biol.,  66,  p.  1)72. 
■    '  This  reagent  (ontains  ,^  .5  grams  of  o-nilroijlieiiyljjropiol'K   ik  i<l  and  5  (  .(  .  of  a  freshly 
preparer]  10  per  c  enl  solution  of  sodium  hydroxide  jjer  liter. 


CARBOHYDRATES. 


25 


7.  Precipitation  by  Alcohol. — To  10  c.c.  of  95  per  cent  alcohol 
add  about  2  c.c.  of  dextrose  solution.  Compare  the  result  with  that 
obtained  under  Dextrin,  7,  page  48. 

8.  Iodine  Test. — Make  the  regular  iodine  test  as  given  under 
Starch,  5,  page  45,  and  keep  this  result  in  mind  for  comparison  with 
the  results  obtained  later  with  starch  and  with  dextrin. 

9.  Diffusibility  of  Dextrose. — Test  the  diffusibility  of  dextrose 
solution  through  animal  membrane,  or  parchment  paper,  making  a 
dialyzer  like  one  of  the  models  shown  in  Fig.  i. 

10.  Moore's  Test. — To  2-3  c.c.  of  sugar  solution  in  a  test-tube 
add  an  equal  volume  of  concentrated  KOH  or  NaOH,  and  boil.  The 
solution  darkens  and  finally  assumes  a  brown  color.     At  this  point  the 


Fig.  I. — DiALYziNG   Appar.\tus  for  Students'  Use. 


odor  of  caramel  may  be  detected.  This  is  an  exceedingly  crude  test 
and  is  of  little  practical  value.  The  brown  color  is  due  to  the  oxida- 
tion of  the  dextrose  and  the  resulting  formation  of  the  potassium  or 
sodium  salts  of  certain  organic  acids  which  are  formed  as  oxidation 
products. 

II.  Reduction  Tests. — To  their  aldehyde  or  ketone  structure 
many  sugars  owe  the  property  of  readily  reducing  alkaline  solutions 
of  the  oxides  of  metals  like  copper,  bismuth  and  mercury;  they  also 
possess  the  property  of  reducing  ammoniacal  silver  solutions  with  the 
separation  of  metallic  silver.  Upon  this  property  of  reduction  the 
most  widely  used  tests  for  sugars  are  based.  When  whitish-blue  cu- 
pric  hydroxide  in  suspension  in  an  alkaline  liquid  is  heated  it  is  con- 
verted into  insokible  black  cupric  oxide,  but  if  a  reducing  agent  like 
certain  sugars  be  present  the  cupric  hydroxide  is  reduced  to  insoluble 
yellow  cuprous  hydroxide,  which  in  turn,  on  further  heating,  may  be 


26  PHYSIOLOGICAL   CHEMISTRY. 

converted  into  brownish-red  or  red  cuprous  oxide.     These  changes 
are  indicated  as  follows: 

OH 

Cu  —  Cu=0  +  H,0. 

\  Cupric  oxide 

OH 

Cupric  hydroxide 
(whitish-blue) . 

OH 

/ 
Cu 

\ 
OH 


—  2Cu-tOH  +  H,0+0. 

•H 

/ 


CW\  Cuprous  hydroxide 

^^  (yeUow). 


Cu 


Cu 

\ 
OH 

Cu— OH  \ 

I  —  0  +  H.O. 

Cu— OH  \  /    • 

Cu 

Cuprous  hydroxide  Cuprous  oxide 

(yellow).  (brownish-red). 

The  chemical  equations  here  discussed  are  exemplified  in  Trom- 
mer's  and  Fehling's  tests. 

(a)  Trommer's  Test. — To  5  c.c.  of  sugar  solution  in  a  test-tube 
add  one-half  its  volume  of  KOH  or  NaOH.  Mix  thoroughly  and 
add,  drop  by  drop,  a  very  dilute  solution  of  cupric  sulphate.  Con- 
tinue the  addition  until  there  is  a  slight  permanent  precipitate  of  cupric 
hydroxide  and  in  consequence  the  solution  is  slightly  turbid.  Heat, 
and  the  cupric  hydroxide  is  reduced  to  yellow  cuprous  hydroxide  or 
to  brownish-red  cuprous  oxide.  If  the  solution  of  cupric  sulphate 
used  is  too  strong  a  small  brownish-red  precipitate  produced  in  a 
weak  sugar  solution  may  be  entirely  masked.  On  the  other  hand, 
particularly  in  testing  for  sugar  in  the  urine,  if  too  little  cupric  sul- 
phate is  used  a  light-colored  precipitate  formed  by  uric  acid  and  purine 
bases  may  obscure  the  brownish-red  precipitate  of  cuprous  oxide. 
The  action  of  KOH  or  NaOH  in  the  presence  of  an  excess  of  sugar 
and  insufficient  copper  will  produce  a  brownish  color.     Phosphates 


CARB  OHYDRATES .  2  7 

of  the  alkaline  earths  may  also  be  precipitated  in  the  alkaline  solution 
and  be  mistaken  for  cuprous  hydroxide.  Trommer's  test  is  not  very- 
satisfactory. 

(b)  Fehling's  Test. — To  about  i  c.c.  of  Fehling's  solution*  in  a 
test-tube  add  about  4  c.c.  of  water,  and  boil.  This  is  done  to  deter- 
mine whether  the  solution  will  of  itself  cause  the  formation  of  a  pre- 
cipitate of  brownish-red  cuprous  oxide.  If  such  a  precipitate  forms, 
the  Fehling's  solution  must  not  be  used.  Add  sugar  solution  to  the 
warm  Fehling's  solution  a  few  drops  at  a  time  and  heat  the  mixture 
after  each  addition.  The  production  of  yellow  cuprous  hydroxide 
or  brownish-red  cuprous  oxide  indicates  that  reduction  has  taken 
place.  The  yellow  precipitate  is  more  likely  to  occur  if  the  sugar 
solution  is  added  rapidly  and  in  large  amount,  whereas  with  a  less 
rapid  addition  of  smaller  amounts  of  sugar  solution  the  brownish- 
red  precipitate  is  generally  formed. 

This  is  a  much  more  satisfactory  test  than  Trommer's,  but  even 
this  test  is  not  entirely  reliable  when  used  to  detect  sugar  in  the  urine. 
Such  bodies  as  conjugate  glycuronates,  uric  acid,  nucleo-protein  and 
homogentisic  acid  when  present  in  sufficient  amount  may  produce  a 
result  similar  to  that  produced  by  sugar.  Phosphates  of  the  alkaline 
earths  may  be  precipitated  by  the  alkali  of  the  Fehling's  solution  and 
in  appearance  may  be  mistaken  for  cuprous  hydroxide.  Cupic  hy- 
droxide may  also  be  reduced  to  cuprous  oxide  and  this  in  turn  be 
dissolved  by  creatinine,  a  normal  urinary  constituent.  This  will 
give  the  urine  under  examination  a  greenish  tinge  and  may  obscure 
the  sugar  reaction  even  when  a  considerable  amount  of  sugar  is  present. 

(c)  Benedict's  Modifications  of  Fehling's  Test. — First  Modificatiofi. — 
To  2  c.c.  of  Benedict's  solution^  in  a  test-tube  add  6  c.c.  of  distilled 
water  and  7-9  drops  (not  more)  of  the  solution  under  examination. 
Boil  the  mixture  vigorously  for  about  15-30  seconds  and  permit  it 

'Fehling's  solution  is  composed  of  two  definite  solutions— -a  cupric  sulphate  solution 
and  an  alkaline  tartrate  solution,  which  may  be  prepared  as  follows: 

Cupric  sulphate  solu(io7t  =  T,4..6i,  grams  of  cupric  sulphate  dissolved  in  water  and  made 
up  to  500  c.c. 

Alkaline  tartrate  solution  =  125  grams  of  potassium  hydroxide  and  173  grams  of  Rochelle 
salt  dissolved  in  water  and  made  up  to  500  c.c. 

These  solutions  should  be  preserved  separately  in  rubber-stoppered  bottles  and  mixed 
in  equal  volumes  when  needed  for  use.     This  is  done  to  prevent  deterioration. 

-  Benedict's  modified  Fehling  solution  consists  of  two  definite  solutions — a  cupric 
sulphate  solution  and  an  alkaline  tartrate  solution,  which  may  be  prepared  as  follows: 

Cupric  sulphate  solution  =24 -6^  grams  of  cupric  sulphate  dissolved  in  water  and  made 
up  to  500  c.c. 

Alkaline  tartrate  solution  =  100  grams  of  anhydrous  sodium  carbonate  and  173  grams  of 
Rochelle  salt  dissolved  in  water  and  made  up  to  t;oo  c.c. 

These  solutions  should  be  preserved  separately  in  rubber-stoppered  bottles  and  mixed 
in  equal  volumes  when  needed  for  use.     This  is  done  to  prevent  deterioration. 


28  PHYSIOLOGICAL    CHEMISTRY. 

to  cool  to  room  temperature  spontaneously.  (If  desired  this  proc- 
ess may  be  repeated,  although  it  is  ordinarily  unnecessary.)  If  sugar 
is  present  in  the  solution  a  precipitate  will  form  which  is  often  bluisJi- 
green  or  greeii  at  first,  especially  if  the  percentage  of  sugar  is 
low,  and  which  usually  becomes  yellowish  upon  standing.  If  the 
sugar  present  exceeds  0.06  per  cent  this  precipitate  generally  forms 
at  or  below  the  boiling  point,  whereas  if  less  than  0.06  per  cent  of 
sugar  is  present  the  precipitate  forms  more  slowly  and  generally 
only  after  the  solution  has  cooled. 

Benedict  claims,  whereas  the  original  Fehling  test  will  not  serve 
to  detect  sugar  when  present  in  a  concentration  of  less  than  0.1 
per  cent,  that  the  above  modification  will  serve  to  detect  sugar 
when  present  in  as  small  quantity  as  0.015-0.02  per  cent. 

The  modified  Fehling  solution  used  in  the  above  test  differs  from 
the  original  Fehling  solution  in  that  100  grams  of  sodium  carbonate 
is  substituted  for  the  125  grams  of  potassium  hydroxide  ordinarily  used, 
thus  forming  a  Fehling  solution  which  is  considerably  less  alkaline 
than  the  original.  This  alteration  in  the  composition  of  the  Fehling 
solution  is  of  advantage  in  the  detection  of  sugar  in  the  urine  inas- 
much as  the  strong  alkalinity  of  the  ordinary  Fehling  solution  has  a 
tendency,  when  the  reagent  is  boiled  with  a  urine  containing  a  small 
amount  of  dextrose,  to  decompose  sufficient  of  the  sugar  to  render 
the  detection  of  the  remaining  portion  exceedingly  difficult  by  the 
usual  technic.  Benedict  claims  that  for  this  reason  the  use  of  his 
modified  solution  permits  the  detection  of  much  smaller  amounts  of 
sugar  than  does  the  use  of  the  ordinary  Fehling  solution.  He  has 
further  modified  his  solution  for  use  in  the  quantitative  determina- 
tion of  sugar  (see  Chapter  XXII). 

Second  Modification} — Very  recently  Benedict  has  further  modi- 
fied his  solution  and  has  succeeded  iin  obtaining  one  which  does  not 
deteriorate  upon  long  standing.^  The  following  is  the  procedure 
for  the  detection  of  dextrose  in  solution:  To  five  cubic  centimeters 
of  the  reagent  in  a  test-tube  add  eight  (not  more)  drops  of  the  solu- 

'  Private  <fjmmuni(  ation  from  \)r.  S.  R.  Uencdic  t. 

'  r^cncdiri's  new  solution  has  the  followinj^  composition: 

Cufjric  sulphate '7-3  j^nims. 

.Soflium  citrate '  73  o  grams. 

Soflium  rarhonale  (anhydrous) loo.o  grams. 

Dislillefl  water  to  make  i  liter. 
With  the  aid  of  licat  dissolve  the  .sodium  citrate  and  rarlionate  in  aiioul  600  c.c.  of 
water.  Pour  (through  a  folded  filler  |ja|)er  if  nec;es.sary)  into  a  glass  graduate  and  make 
up  to  850  c.c.  Dissolve  the  tupric  sul[)hale  in  about  loo  c.c.  of  water  and  make  up  to 
r5o  c.c.  Pour  the  f.arhonatc-(  ilrale  solution  into  a  large  beaker  or  casserole  and  add  the 
cupric  sulphate  solution  slowly,  with  constant  stirring.  The  mixed  solution  is  ready  for 
iis<;  and  df)cs  not  deteriorate  upon  long  standing. 


CARBOHYDRATES.  29 

tion  under  examination.  Boil  the  mixture  vigorously  for  from  one 
to  two  minutes  and  then  allow  the  fluid  to  cool  spontaneously.  In 
the  presence  of  dextrose  the  entire  body  of  the  solution  will  he  filled  with 
a  precipitate,  which  may  be  red,  yellow  or  green  in  color,  depending 
upon  the  amount  of  sugar  present.  If  no  dextrose  is  present,  the 
solution  will  remain  perfectly  clear.  (If  urine  is  being  tested,  it 
may  show  a  very  faint  turbidity,  due  to  precipitated  urates.)  Even 
very  small  quantities  of  dextrose  (o.i  per  cent)  yield  precipitates  of 
surprising  bulk  with  this  reagent,  and  the  positive  reaction  for  dex- 
trose is  the  filling  of  the  entire  body  of  the  solution  with  a  precipitate, 
so  that  the  solution  becomes  opaque.  Since  amount  rather  than 
color  of  the  precipitate  is  made  the  basis  of  this  test,  it  may  be  applied 
even  for  the  detection  of  small  quantities  of  dextrose,  as  readily  in 
artificial  light  as  in  daylight. 

{d)  Boettger's  Test. — To  5  c.c.  of  sugar  solution  in  a  test-tube 
add  I  c.c.  of  KOH  or  NaOH  and  a  very  small  amount  of  bismuth 
subnitrate,  and  boil.  The  solution  will  gradually  darken  and  finally 
assume  a  black  color  due  to  reduced  bismuth.  If  the  test  is  made 
on  urine  containing  albumin  this  must  be  removed,  by  boiling  and 
filtering,  before  applying  the  test,  since  with  albumin  a  similar  change 
of  color  is  produced  (bismuth  sulphide). 

(e)  Nylander's  Test  {Almen's  Test). — To  5  c.c.  of  sugar  solu- 
tion in  a  test-tube  add  one-tenth  its  volume  of  Nylanders  reagent^' 
and  heat  for  five  minutes  in  a  boiling  water-bath.'  The  solution 
will  darken  if  reducing  sugar  is  present,  and  upon  standing  for  a  few 
moments  a  black  color  will  appear.  This  color  is  due  to  the  pre- 
cipitation of  bismuth.  If  the  test  is  made  on  urine  containing  albumin 
this  must  be  removed,  by  boiling  and  filtering,  before  applying  the 
test,  since  with  albumin  a  similar  change  of  color  is  produced.  Dex- 
trose when  present  to  the  extent  of  0.08  per  cent  may  be  detected  by 
this  reaction.  It  is  claimed  by  Bechold  that  Nylander's  and  Boettger's 
tests  give  a  negative  reaction  with  solutions  containing  sugar  when 
mercuric  chloride  or  chloroform  is  present.  Other  observers^  have 
failed  to  verify  the  inhibitory  action  of  mercuric  chloride  and  have 
shown  that  the  inhibitory  influence  of  chloroform  may  be  overcome  by 

^  Nylander's  reagent  is  prepared  by  digesting  2  grams  of  bismuth  sul^nitrate  and  4 
grams  of  Rochelle  salt  in  100  c.c.  of  a  10  per  cent  potassium  liydroxide  solution.  The 
reagent  is  then  cooled  and  filtered. 

2  Hamm  irsten  suggests  that  the  mixture  should  be  boiled  2-5  minutes  (according  to  the 
sugar  content)  over  a  free  flame  and  the  tube  then  permitted  to  stand  5  minutes  before 
drawing  conclusions. 

'  Rehfuss  and  Hawk;  Journal  of  Biological  Chemistry,  \'II,  p.  267,  igio:  also  Zeidlitz: 
Upsala  Lakareforen  Fork.,  N.  F.,  XI,  1906. 


30  PHYSIOLOGICAL    CHEMISTRY. 

raising  the  temperature  of  the  urine  to  the  boiHng  point  for  a  period  of 
five  minutes  previous  to  making  the  test.  Urines  rich  in  indican,  uro- 
chrome,  uroerythrin  or  hcemaio por phyrin,  as  well  as  urines  excreted  after 
the  ingestion  of  large  amounts  of  certain  medicinal  substances,  may 
give  a  darkening  of  Nylander's  reagent  similar  to  that  of  a  true  sugar 
reaction.  It  is  a  disputed  point  whether  the  urine  after  the  adminis- 
tration of  urotropin  will  reduce  Nylander's  reagent.^ 

According  to  Rustin  and  Otto,  the  addition  of  PtCl^  increases 
the  delicacy  of  Nylander's  reaction.  They  claim  that  this  procedure 
causes  the  sugar  to  be  converted  quantitatively.  No  quantitative 
method  has  yet  been  devised,  howe\'er,  based  upon  this  principle. 

Bohmansson-  before  testing  the  urine  under  examination  treats 
it  (lo  c.c.)  with  1/5  volume  of  25  per  cent  hydrochloric  acid  and 
about  1/2  volume  of  Ijone  black.  This  mixture  is  shaken  one  minute, 
then  filtered  and  the  neutralized  filtrate  tested  by  Nylander's  reaction. 
Bohmansson  claims  that  this  procedure  removes  certain  interfering 
substances,  in  particular  urochrome. 

A  positive  Nylander  or  Boettger  test  is  probably  due  to  the  fol- 
lowing reactions: 

(a)  Bi(OH)3N03  4-KOH  =  Bi(OH)3-^KN03. 

(b)  2Bi(OH)3-30  =  Bi3-f3H30. 

12.  Fermentation  Test. — "Rub  up"  in  a  mortar  about  20  c.c. 
of  the  sugar  solution  with  a  small  piece  of  compressed  yeast.  Transfer 
the  mixture  to  a  saccharomcter  (shown  in  Fig.  2,  p.  31)  and  stand  it  aside 
in  a  warm  place  for  about  twelve  hours.  If  the  sugar  is  fermentable, 
alcoholic  fermentation  will  occur  and  carbon  dioxide  will  collect  as  a 
gas  in  the  upper  portion  of  the  tulK\  On  the  completion  of  ferment- 
ation introduce  a  little  potassium  hydroxide  solution  into  the  grad- 
uated portion  by  means  of  a  bent  pipette,  place  the  thumb  tightly 
over  the  opening  in  the  apparatus  and  in\'ert  the  saccharometer. 
Explain  the  result. 

13.  Barfoed's  Test.  Place  about  5  c.c.  of  JJarfoed's  solution^  in 
a  test-tube  and  heat  lo  boih'ng.  Add  dextrose  solution  slowly,  a  few 
drops  at  a  time,  heating  after  each  addition.  Reduction  is  indicated 
by  the  formation  of  a  red  ])re(ipitate.     If  the  jjrecipitate  does  not 

'  Abl;  Arrhives  <>/  I'rdintrirs,  XXIV,  p.  275,  1007;  also  VVciihicc  hi ;  Schweiz.  Wochschr., 
XLVU,  p.  577,  1909. 

'■'Bohmansson:  Biockem.  Zeil.,  79,  |j.  28r. 

'  Barfoed's  solution  is  preparcfi  as  follows:  Dissolve  4.5  grams  of  neulral  crystallized 
<  upri<  a<etat(  in  100  c.c.  of  water  anrl  add  i ,  2  c.c.  of  50  per  cent  acetic  ai  id. 


CARBOHYDRATES. 


31 


form  upon  continued  boiling  allow  the  tube  to  stand  a  few 
minutes  and  examine.  Sodium  chloride  interferes  with  the  reac- 
tion  (Welker). 

Barfoed's  test  is  not  a  specific  test  for  dextrose  as  is  frequently  stated, 
but  simply  serves  to  detect  monosaccharides.  Disaccharides  will  also 
respond  to  the  test,  according  to  Hinkel  and  Sherman,  if  the  sugar 
solution  is  boiled  sufficiently  long,  in  contact  with  the  reagent,  to  hydro- 
lyze  the  disaccharide  through  the  action  of  the 
acetic  acid  present  in  the  Barfoed's  solution. 

14.  Formation  of  Caramel. — Gently  heat 
a  small  amount  of  pulverized  dextrose  in  a 
test-tube.  After  the  sugar  has  melted  and 
turned  brown,  allow  the  tube  to  cool,  add 
water  and  warm.  The  coloring  matter  pro- 
duced is  known  as  caramel. 

15.  Demonstration  of  Optical  Activity. — 
A  demonstration  of  the  use  of  the  polariscope, 
by  the  instructor,  each  student  being  required 
to  take  readings  and  compute  the  "specific 
rotation." 

Use  of  the  Polariscope. 

For  a  detailed  description  of  the  different 
forms  of  polariscopes,  the  method  of  manipula-  cHAROiiETER. 

tion  and  the  principles  involved,  the  student 

is  referred  to  any  standard  text-book  of  physics.  A  brief  descrip- 
tion follows:  An  ordinary  ray  of  light  vibrates  in  every  direction. 
If  such  a  ray  is  caused  to  pass  through  a  "polarizing"  Nicol  prism 
it  is  resolved  into  two  rays,  one  of  which  vibrates  in  every  direction  as 
before  and  a  second  ray  which  vibrates  in  ane  plane  only.  This  latter 
ray  is  said  to  be  polarized.  Many  organic  substances  (sugars,  proteins, 
etc.)  have  the  power  of  twisting  or  rotating  this  plane  of  polarized  light, 
the  extent  to  which  the  plane  is  rotated  depending  upon  the  number 
of  molecules  which  the  polarized  light  passes.  Substances  which  pos- 
sess this  power  are  said  to  be  "optically  active."  The  specific  rotation 
of  a  substance  is  the  rotation  expressed  in  degrees  which  is'  afforded  by 
one  gram  of  substance  dissolved  in  i  c.c.  of  water  in  a  tube  one  deci- 
meter in  length.  The  specific  rotation,  (q:)^,  may  be  calculated  by 
means  of  the  following  formula. 


32  PHYSIOLOGICAL    CHEMISTRY. 

a 

p.l 

in  which 

D  =  sodium  light. 

a  =  observed  rotation  in  degrees. 

/>  =  grams  of  substance  dissolved  in  i  c.c.  of  liquid. 

/  =  length  of  the  tube  in  decimeters. 
If  the  specific  rotation  has  been  determined  and  it  is  desired  to  ascertain 
the  per  cent  of  the  substance  in  solution,  this  may  be  obtained  by 
the  use  of  the  following  formula, 

a 

The  value  of  p  multiplied  by  loo  will  be  the  percentage  of  the  sub- 
stance in  solution. 

An  instrument  by  means  of  which  the  extent  of  the  rotation  may 
be  determined  is  called  a  polariscope  or  polarimeler.     Such  an  instru- 


FiG.  3. — One  Form  ok  Laurent  Polariscopk. 

n,  MUrosropv  for  reafling  the  scale;  (',  a  vt-rnier;  E,  jjosition  of  the  :inaly/.inf(  Nicx)l  piisni; 

//,  polarizing  Nitol  piism  in  llu-  lu!)i'  Ix-low  lliis  point. 


ment  designed  especially  for  the  examination  of  sugar  .solutions  is 
termed  a  saccharimeler  or  polar izini;  saccharimeter.  The  form  ol 
j>oIariscope  in  Fig.  3,  above,  consists  essentially  of  a  long  barrel  pro- 
vided with  a  Nicol  prism  at  either  end  (Fig.  4,  p.  33).  The  solution 
under  examination  is  contained  in  a  tube  which  is  ])laced  between  these 
two  prisms.  At  the  front  end  of  the  instrument  is  an  adjusting  eye- 
piece for  focusing  and  a  large  recording  disc  which  registers  in  degrees 
and  fractions  of  a  degree.  The  light  is  admitted  into  the  far  end  of 
the  instrument  and  is  j)ol;iri/,cfl  hy  jjassing  through  a  Nicol  prism.     This 


CARBOHYDRATES. 


33 


polarized  ray  then  traverses  the  column  of  liquid  within  the  tube 
mentioned  above  and  if  the  substance  is  optically  active  the  plane  of 
the  polarized  ray  is  rotated  to  the  right  or  left.     Bodies  rotating  the 


Fig.  4. — Diagrammatic  Representation  of  the  Course  of  the  Light  through  the 
Laurent  Polariscope.  (The  direction  is  reversed  from  that  of  Fig.  3,  p.  32-) 
a,  Bichromate  plate  to  purify  the  light;  h,  the  polarizing  Nicol  prism;  c,  a  thin  quartz 
plate  covering  one-half  the  field  and  essential  in  producing  a  second  polarized  plane;  d, 
tube  to  contain  the  liquid  under  examination;  e,  the  analyzing  Nicol  prism;/ and  g,  ocular 
lenses. 

ray  to  the  right  are  called  dextro-rotatory  and  those  rotating  it  to  the 
left  Icevo-rotatory. 

Within  the  apparatus  is  a  disc  which  is  so  arranged  as  to  be  without 


Fig.  5. — ^Polariscope  (Schmidt  and  Haensch  Model). 

lines  and  uniformly  light  at  zero.  Upon  placing  the  optically  acti\e 
substance  in  position,  however,  the  plane  of  polarized  light  is  rotated 
or  turned  and  it  is  necessary  to  rotate  the  disc  through  a  certain  number 


34  PHYSIOLOGICAL   CHEMISTRY. 

of  degrees  in  order  to  secure  the  normal  conditions,  /.  e.,  'Svithout  lines 
and  uniformly  light."  The  difference  between  this  reading  and  the 
zero  is  a  or  the  observed  rotation  in  degrees. 

Polarizing  saccharimeters  are  also  constructed  by  which  the  per- 
centage of  sugar  in  solution  is  determined  by  making  an  observation 
and  multiplying  the  value  of  each  division  on  a  horizontal  sliding 
scale  by  the  value  of  the  division  expressed  in  terms  of  dextrose.  This 
factor  may  vary  according  to  the  instrument. 

CH.OH 
L^VULOSE,  (CHOH)3. 

I 

CO 

I 
CH,OH 

As  already  stated,  laevulose,  sometimes  called  fructose  or  fruit 
sugar,  occurs  widely  disseminated  throughout  the  plant  kingdom 
in  company  with  dextrose.  Its  reducing  power  is  somewhat  weaker 
than  that  of  dextrose.  Liievulose  does  not  ordinarily  occur  in  the 
urine  in  diabetes  mellitus,  but  has  been  found  in  exceptional  cases. 
With  phenylhydrazine  it  forms  the  same  osazone  as  dextrose.  With 
methylphenylhydrazinc,  laevulose  forms  a  characteristic  methyl- 
phcnyllaevulosazonc. 

(For  a  further  discussion  of  Iccvulosc  see  the  section  on  Hexoscs, 

p.   21.) 

Experiments  on  L^vulose. 

1-T3.  Repeat  these  experiments  as  given  under  Dextrose,  pages 
22-30. 

14.  Seliwanoff's  Reaction.-- To  5  c.c.  of  Seliwanoff's  reagent^ 
in  a  test-tube  add  a  few  drops  of  a  laevulose  solution  and  heat  the 
mixture  to  boiling.  A  positive  reaction  is  indicated  by  the  production 
of  a  red  color  and  the  sej)aration  of  a  red  ])recipitate.  The  latter  may 
be  dissolved  in  alcohol  to  which  it  will  imjjarl  a  striking  red  color. 

If  the  boiling  be  prolonged  a  similar  reaction  may  be  o])taine(l 
with  solutions  of  dextrose  or  maltose. 

15.  Borchardt's  Reaction.  -  To  about  5  c.c.  of  a  solution  of 
laevulose  in  a  test-tube  add  an  equal   volume  of  25  per  cent  hydro 

'  .Seliwanoff's  reagent  may  Ijc  |)rei>iircd  Ijy  dissolving  0.05  gram  of  resorcin  in  100  c.r. 
of  dilute  (1:2)  hydrochloric  arid. 


CARBOHYDRATES-  35 

chloric  acid  and  a  few  crystals  of  resorcin.  Heat  to  boiling  and  after 
the  production  of  a  red  color,  cool  the  tube  under  running  water  and 
transfer  to  an  evaporating  dish  or  beaker.  Make  the  mixture  slightly 
alkaline  with  solid  potassium  hydroxide,  return  it  to  a  test-tube,  add 
2-3  c.c.  of  acetic  ether  and  shake  the  tube  vigorously.  In  the  presence 
of  laevulose,  the  acetic  ether  is  colored  yellow.  (For  further  discussion 
of  the  test  see  Chapter  XIX.) 

16.  Formation  of  Methylphenyllaevulosazone. — To  a  solution 
of  1.8  grams  of  laevulose  in  lo  c.c.  of  water  add  4  grams ^  of  methyl- 
phenylhydrazine  and  enough  alcohol  to  clarify  the  solution.  Intro- 
duce 4  c.c  of  50  per  cent  acetic  acid  and  heat  the  mixture  for  5-10 
minutes  on  a  boiling  water-bath.^  On  standing  15  minutes  at  room 
temperature,  crystallization  begins  and  is  complete  in  two  hours. 
By  scratching  the  sides  of  the  flask  or  by  inoculation,  the  solution 
quickly  congeals  to  form  a  thick  paste  of  reddish-yellow  silky  needles. 
These  are  the  crystals  of  methylphenyllavidosazone.  They  may  be 
recrystallized  from  hot  95  per  cent  alcohol  and  melt  at  153°  C. 

CH^OH 
GALACTOSE,  (CHOH),. 

I 

CHO 

Galactose  occurs  with  dextrose  as  one  of  the  products  of  the 
hydrolysis  of  lactose.  It  is  dextro-rotatory,  forms  an  osazone  with 
phenylhydrazine  and  ferments  slowly  with  yeast.  Upon  oxidation 
with  nitric  acid  galactose  yields  mucic  acid,  thus  differentiating  this 
monosaccharide  from  dextrose  and  laevulose.  Lactose  also  yields 
mucic  acid  under  these  conditions.  The  mucic  acid  test  may  be  used 
in  urine  examination  to  differentiate  lactose  and  galactose  from  other 
reducing  sugars. 

Experiments  on  Galactose. 

I.  Tollens'  Reaction. — To  equal  volumes  of  galactose  solution 
and  hydrochloric  acid  (sp.  gr.  1.09)  add  a  little  phloroglucin,  and 
heat  the  mixture  on  a  boiling  water-bath.  Galactose,  pentose  and 
glycuronic  acid  will  be  indicated  by  the  appearance  of  a  red  color. 
Galactose  may  be  differentiated  from  the  two  latter  substances  in 

^  3.66  grams  if  absolutely  pure. 
^  Longer  heating  is  to  be  avoided. 


36  PHYSIOLOGICAL    CHEMISTRY. 

that  its  solutions  exhibit  no  absorption  bands  upon  spectroscopical 
examination. 

2.  Mucic  Acid  Test. — Treat  100  c.c.  of  the  solution  containint;; 
galactose  with  20  c.c.  of  concentrated  nitric  acid  (sp.  gr.  1.4)  and 
evaporate  the  mixture  in  a  broad,  shallow  glass  vessel  on  a  boiling 
water-bath  until  the  volume  of  the  mixture  has  been  reduced  to 
about  20  c.c.  At  this  point  the  fluid  should  be  clear,  and  a  fine  white 
precipitate  of  mucic  acid  should  form.  If  the  percentage  of  galactose 
present  is  low  it  may  be  necessary  to  cool  the  solution  and  permit 
it  to  stand  for  some  time  before  the  precipitate  will  form.  It  is  im- 
possible to  differentiate  between  galactose  and  lactose  by  this  test, 
but  the  reaction  serves  to  differentiate  these  two  sugars  from  all 
other  reducing  sugars.  Differentiate  lactose  from  galactose  by  means 
of  Barfoed's  test  (p.  30). 

3.  Phenylhydrazine  Reaction. — Make  the  test  according  to 
directions  given  under  Dextrose,  3  or  4,  pages  23  and  24. 

Pentoses,  C^Hj^Os. 

In  plants  and  more  particularly  in  certain  gums,  very  complex 
carbohydrates,  called  pentosans,  occur.  These  pentosans  through 
hydrolysis  by  acids  may  be  transformed  into  pentoses.  Pentoses 
do  not  ordinarily  occur  in  the  animal  organism,  but  have  been  found 
in  the  urine  of  morphine  habitues  and  others,  their  occurrence  some- 
times being  a  persistent  condition  without  known  cause.  They  are 
'non-fermentable,  have  strong  reducing  power  and  form  osazones 
with  phenylhydrazine.  Pentoses  are  an  important  constituent  of 
the  dietary  of  herbivorous  animals.  Glycogen  is  said  to  be  formed 
after  the  ingestion  of  these  sugars  containing  five  oxygen  atoms. 
This,  however,  has  not  been  conclusively  proven.  On  distillation 
with  strong  hydrochloric  acid  pentoses  and  pentosans  yield  furfurol, 
which  can  be  flelected  by  its  characteristic  red  reaction  with  aniline- 
acetate   paper. 

CH.OH 

I 

ARABINOSE,  (CHOH)3. 

I 

CHO 

Arabinose  is  one  of  the  most  important  of  the  pentoses.  The 
/  arabinose  may  be  obtained  from  gum  arabif,  phim  or  cherry  gum 


CARBOHYDRATES.  37 

by  boiling  for  several  hours,  with  1-2  per  cent  sulphuric  acid.  This 
pentose  is  dextro-rotatory,  forms  an  osazone  and  has  reducing  power. 
The  «-arabinose  has  been  isolated  from  the  urine  and  yields  an  osazone 
which  melts  at  i66°-i68°  C. 


Experiments  on  Arabinose. 

1.  Tollens'  Reaction. — To  equal  volumes  of  arabinose  solu- 
tion and  hydrochloric  acid  (sp.  gr.  1.09)  add  a  little  phloroglucin 
and  heat  the  mixture  on  a  boiling  water-bath.  Galactose,  pentose 
or  glycuronic  acid  will  be  indicated  by  the  appearance  of  a  red  color. 
To  differentiate  between  these  bodies  make  a  spectroscopic  examina- 
tion and  look  for  the  absorption  band  between  D  and  E  given  by  pen- 
toses and  glycuronic  acid.  Differentiate  between  the  two  latter 
bodies  by  the  melting-points  of  their  osazones. 

Compare  the  reaction  with  that  obtained  with  galactose  (page  35). 

2.  Orcin  Test. — Repeat  i,  using  orcin  instead  of  phloroglucin. 
A  succession  of  colors  from  red  through  reddish-blue  to  green  is 
produced.  A  green  precipitate  is  formed  which  is  soluble  in  amyl 
alcohol  and  has  absorption  bands  between  C  and  D. 

3.  Phenylhydrazine  Reaction. — Make  this  test  on  the  arabinose 
solution  according  to  directions  given  under  Dextrose,  3  or  4,  pages 
23  and  24. 

CH,OH 

XYLOSE,   (CHOH)3. 

CHO 

Xylose,  or  wood  sugar,  is  obtained  by  boiling  wood  gums  with 
dilute  acids  as  explained  under  Arabinose,  page  36.  It  is  dextro- 
rotatory and  forms  an  osazone. 

Experiments  on  Xylose. 

1-3.  Same  as  for  arabinose  (see  above). 

RHAMNOSE,    C.Hj^Oj. 

Rhamnose  or  methyl-pentose  is  an  example  of  a  true  carbohydrate 
which  does  not  have  the  H  and  O  atoms  present  in  the  proportion 


38  PHYSIOLOGICAL   CHEMISTRY. 

to  form  water.  Its  formula  is  C6Hj205.  It  has  been  found  that 
rhamnose  when  ingested  by  rabbits  or  hens  has  a  positive  influence 
upon  the  formation  of  glycogen  in  those  organisms. 

DISACCHARIDES,  C,,I{,,0^,. 

The  disaccharides  as  a  class  may  be  divided  into  two  rather  dis- 
tinct groups.  The  first  group  would  include  those  disaccharides 
which  are  found  in  nature  as  such,  e.  g.,  sucrose  and  lactose  and  the 
second  group  would  include  those  disaccharides  formed  in  the  hy- 
drolysis of  more  complex  carbohydrates,  e.  g.,  maltose,  and  iso-maltose. 

The  disaccharides  have  the  general  formula  CjjH.,,©^,  to  which, 
in  the  process  of  hydrolysis,  a  molecule  of  water  is  added  causing 
the  single  disaccharide  molecule  to  split  into  two  monosaccharide 
(hexose).  molecules.  The  products  of  the  hydrolysis  of  the  more 
common  disaccharides  are  as  follows: 

Maltose  =  dextrose  +  dextrose. 
Lactose  =  dextrose -[-galactose. 
Sucrose  =  dextrose -hlaevulose. 

All  of  the  more  common  disaccharides  except  sucrose  have  the 
power  of  reducing  certain  metallic  oxides  in  alkaline  solution,  notably 
those  of  copper  and  bismuth.  This  reducing  power  is  due  to  the 
presence  of  the  aldehyde  group  ( — CHO)  in  the  sugar  molecule. 

MALTOSE,    C^H^O,,. 

Maltose  or  malt  sugar  is  formed  in  the  hydrolysis  of  starch  through 
the  action  of  an  enzyme,  vegetable  amylase  (diastase),  contained  in 
sprouting  barley  or  malt.  Certain  enzymes  in  the  saliva  and  in  the 
];ancreatic  juice  may  also  cause  a  similar  hydrolysis.  Maltose  is  also 
an  intermediate  product  of  the  action  of  dilute  mineral  acids  upon 
starch.  It  is  strongly  dextro-rotatory,  reduces  metallic  oxides  in 
alkaline  solution  and  is  fermentable  by  yeast  after  being  inverted 
(see  Polysaccharides,  page  42)  by  the  enzyme  maltase  of  the  yeast. 
In  common  with  the  other  disaccharides,  maltose  may  be  hydroly/ed 
with  the  formation  of  two  molecules  of  monosaccharide.  In  this  in- 
stance the  j>roducts  are  two  molecules  of  dextrose.  With  y)henylhy- 
drazine  maltose  forms  an  osazone,  maltosazone.  The  following 
formula  represents  the  probable  structure  of  maltose: 


CARB  OHYDRATES . 
CH2OH  CHO 

CHOH  CHOH 

CHO  CHOH 

I  :  I 

CHOH  CHOH 

i  ! 

CHOH  CHOH 

I  I 

C— -^ O— CH, 


H 


Maltose. 


Experiments  on  Maltose.  •  • 

1-13.  Repeat  these  experiments  as  given  under  Dextrose,  pages 
22—30. 

ISO-MALTOSE,    CHO 

Iso-maltose,  an  isomeric  form  of  maltose,  is  formed,  along  with 
maltose,  by  the  action  of  diastase  upon  starch  paste,  and  also  by 
the  action  of  hydrochloric  acid  upon  dextrose.  It  also  occurs  with 
maltose  as  one  of  the  products  of  salivary  digestion.  It  is  dextro- 
rotatory and  with  phenylhydrazine  gives  an  osazone  which  is  char- 
acteristic. Iso-maltose  is  very  soluble  and  reduces  the  oxides  of 
bismuth  and  copper  in  alkaline  solution.  Pure  iso-maltose  is  probably 
only  slightly  fermentable. 

LACTOSE,    CHO 

'       12     22     11* 

Lactose  or  milk  sugar  occurs  ordinarily  only  in  milk,  but  has 
often  been  found  in  the  urine  of  women  during  pregnancy  and  lac- 
tation. It  may  also  occur  in  the  urine  of  normal  persons  after  the 
ingestion  of  unusually  large  amounts  of  lactose  in  the  food.  It  has  a 
strong  reducing  power,  is  dextro-rotatory  and  forms  an  osazone  with 
phenylhydrazine.  Upon  hydrolysis  lactose  yields  one  molecule  of 
dextrose  and  one  molecule  of  galactose. 

In   the   souring   of   milk   the   bacterium   lactis   and   certain   other 


40  PHYSIOLOGICAL    CHEMISTRY. 

micro-organisms  bring  about  lactic  acid  fermentation  by  transform- 
ing the  lactose  of  the  milk  into  lactic  acid, 

H      OH 

H  — C  — C  — COOH, 

I        i 

H      H 

and  alcohol.  This  same  reaction  may  occur  in  the  alimentary  canal 
as  the  result  of  the  action  of  putrefactive  bacteria.  In  the  preparation 
of  kephyr  and  koumyss  the  lactose  of  the  milk  undergoes  alcoholic 
fermentation,  through  the  action  of  ferments  other  than  yeast,  and 
at  the  same  time  lactic  acid  is  produced.  Lactose  and  galatcose  yield 
mucic  acid  on  oxidation  with  nitric  acid.  This  fact  is  made  use  of  in 
urine  analysis  to  facilitate  the  differentiation  of  these  sugars  from  other 
reducing  sugars. 

Lactose  is  not  fermentable  by  pure  yeast. 

Experiments  on  Lactose. 

1-13.  Repeat  these  experiments  as  given  under  Dextrose,  pages 
22-30. 

14.  Mucic  Acid  Test. — Treat  100  c.c.  of  the  solution  containing 
lactose  with  20  c.c.  of  concentrated  nitric  acid  (sp.  gr.  1.4)  and  evapo- 
rate the  mixture  in  a  broad,  shallow  glass  vessel  on  a  boiling  water- 
bath,  until  the  volume  of  the  mixture  has  been  reduced  to  about  20  c.c. 
At  this  point  the  fluid  should  be  clear,  and  a  fine  white  precipitate  of 
mucic  acid  should  form.  If  the  percentage  of  lactose  present  is  low 
it  may  be  necessary  to  cool  the  solution  and  permit  it  to  stand  for 
some  time  before  the  precipitate  will  appear.  It  is  impossible  to 
dilTerentiate  between  lactose  and  galactose  by  this  test,  but  the  reaction 
serves  to  dififerentiate  these  two  sugars  from  all  other  reducing  sugars. 

DifTerentiate  lactose  from  galactose  by  means  of  f5arfoed's  test, 
page  30. 

SUCROSE,  C,. ,11,20,,. 

Sucrose,  al.so  called  .saccharose  or  cane  sugar,  is  one  of  the  most 
important  of  the  sugars  and  occurs  very  extensively  distributed  in 
plants,  particularly  in  the  sugar  cane,  sugar  beet,  sugar  millet  and 
in  certain  jjalms  and  maj^les. 

Sucrose  is  dcxtro  rc^tatory  and  u[)on  hydrolysis,  as  before  men- 
tioned,  the   molecule  of  sucrose  takes  on   a  molecule  of  water  and 


CARBOHYDRATES.  4I 

breaks  down  into  two  molecules  of  monosaccharide.  The  mono- 
saccharides formed  in  this  instance  are  dextrose  and  iaevulose.  This 
is    the    reaction: 

Sucrose.  Dextrose.  L£e\'-ulose. 

This  process  is  called  inversion  and  may  be  produced  by  bacteria, 
enzymes,  and  certain  weak  acids.  After  this  inversion  the  previously 
strongly  dextro-rotatory  solution  becomes  laevo-rotatory.  This  is 
due  to  the  fact  that  the  Iaevulose  molecule  is  more  strongly  laevo- 
rotatory  than  the  dextrose  molecule  is  dextro-rotatory.  The  product 
of  this  inversion  is  called  invert  sugar. 

Sucrose  does  not  reduce  metallic  oxides  in  alkaline  solution  and 
forms  no  osazone  with  phenylhydrazine.  It  is  not  fermentable  directly 
by  yeast,  but  must  first  be  inverted  by  the  enzyme  sucrose  {invertase 
or  invertin)  contained  in  the  yeast.  The  probable  structure  of  sucrose 
may  be  represented  by  the  following  formula.  Note  the  absence  of 
any  true  sugar  group  or  free  ketone  or  aldehyde  group. 

CH2OH  CH.OH 

CHOH  CHO 

CHO  CHOH 

I  I 

CHOH    i      CHOH 

CHOH    ,      C^ 

(>^^   O     CH.,OH 

\ 
H 

Sucrose. 

Experiments  on  Sucrose. 

1-13.  Repeat  these  experiments  according  to  the  directions  given 
under  Dextrose,  pages  22-30. 

14.  Inversion  of  Sucrose. — To  25  c.c.  of  sucrose  solution  in 
a  beaker  add  5  drops  of  concentrated  HCl  and  boil  one  minute. 
Cool  the  solution,  render  alkaline  with  solid  KOH  and  upon  the 
resulting  fluid  repeat  experiments  3  (or  4)  and  11  as  given  under  Dex- 
trose, pages  23-25.     Explain  the  results. 


42  PHYSIOLOGICAL    CHEMISTRY. 

15.  Production  of  Alcohol  by  Fermentation. — Prepare  a 
strong  (10-20  per  cent)  solution  of  sucrose,  add  a  small  amount  of 
egg  albumin  or  commercial  peptone  and  introduce  the  mixture  into 
a  bottle  of  appropriate  size.  Add  yeast,  and  by  means  of  a  bent  tube 
inserted  through  a  stopper  into  the  neck  of  the  bottle,  conduct  the 

escaping  gas  into  water.  As  fermenta- 
tion proceeds  readily  in  a  warm  place 
the  escaping  gas  may  be  collected  in  a 
eudiometer  tube  and  examined.  When 
the  activity  of  the  yeast  has  practically 
ceased,  filter  the  contents  of  the  bottle 
into  a  flask  and  distil  the  mixture. 
Catch,  the  first  portion  of  the  distillate 
separately  and  test  for  alcohol  by  one  of 

,  ,        .  ,  ,     the  following  reactions: 
Fig.  6. — Iodoform.     {Auleririelh.)  ° 

{a)  Iodoform   Test. — Render  2-3  c.c. 

of  the  distillate  alkaline  with  potassium  hydroxide  solution  and  add 

a  few  drops  of  iodine  solution.     Heat  gently  and  note  the  formation 

of  iodoform  crystals.     Examine  these  crystals  under  the  microscope 

and  compare  them  with  those  in  Fig.  6. 

(b)  Aldehyde   Test. — Place    5  c.c.  of  the  distillate  in  a  test-tube, 

add   a  few  drops  of  potassium  dichromate  solution,  K^CrjO^,  and 

render  it  acid  with  dilute  sulphuric  acid.     Boil  the  acid  solution  and 

note  the  odor  of  aldehyde. 

TRISACCHARIDES,    C,,U^,0,,. 

RAFFmOSE. 

This  trisaccharide,  also  called  melitose,  or  melitriose  occurs  in 
cotton  seed,  Australian  manna,  and  in  the  molasses  from  the  prep- 
aration of  beet  sugar.  It  is  dextro-rotatory,  does  not  reduce 
Fehling's  solution,  and  is  only  partially  fermentable  by  yeast. 

Raffinose  may  be  hydrolyzed  by  weak  acids  the  same  as  the  poly- 
saccharides are  hydrolyzed,  the  i)roducts  being  kevulose  and  melil)iose; 
further  hydrolysis  of  the  mulibiose  yields  dextrose  and  galactose. 

POLYSACCHARIDES,  ((„n„/)J,. 

In  general  the  polysaccharides  are  amorjjhous  bodies,  a  few,  how- 
ever, are  crystallizable.  'J'hrough  the  action  of  certain  enzymes  or 
weak  acids  the  polysaccharides  may  be  hydrolyzed  willi  the  formation 
(jf  monosaccharides.     As  a  class  I  lie  polys.'u charidcs  are  (|uite  insolu- 


CARBOHYDRATES.  43 

ble  and  are  non-fermentable  until  inverted.  By  inversion  is  meant 
the  hydrolysis  of  disaccharide  or  polysaccharide  sugars  to  form  mono- 
saccharides, as  indicated  in  the  following  equations: 

(a)  C,,H330,,  +  H30  =  2(C,H,,OJ. 

(b)  C,H,„0,-fH30^C,H,A. 

STARCH,    (C,H,„0,),. 

Starch  is  widely  distributed  throughout  the  vegetable  kingdom, 
occurring  in  grains,  fruits,  and  tubers.  It  occurs  in  granular  form,  the 
microscopical  appearance  being  typical  for  each  individual  starch. 
The  granules,  which  differ  in  size  according  to  the  source,  are  com- 
posed of  alternating  concentric  rings  of  granulose  and  cellulose.  Ordi- 
nary starch  is  insoluble  in  cold  v/ater,  but  if  boiled  with  water  the  cell 
walls  are  ruptured  and  starch  paste  results.  In  general  starch  gives  a 
blue  color  with  iodine. 

Starch  is  acted  upon  by  amylases,  e.  g.,  salivary  amylase  (ptyalin) 
and  pancreatic  amylase  {amylopsin),  with  the  formation  of  soluble 
starch,  erythro-dextrin,  achroo-dextrins,  maltose,  iso-maltose  and  dextrose 
(see  Salivary  Digestion,  page  53).  Maltose  is  the  principal  end-prod- 
uct of  this  enzyme  action.  Upon  boiling  a  starch  solution  with  a 
dilute  mineral  acid  a  series  of  similar  bodies  is  formed,  but  under  these 
conditions  dextrose  is  the  principal  end-product. 

Experiments  on  Starch. 

1.  Preparation  of  Potato  Starch. — Pare  a  raw  potato,  com- 
minute it  upon  a  fine  grater,  mix  with  water,  and  "whip  up"  the 
pulped  material  vigorously  before  straining  it  through  cheese  cloth 
or  gauze  to  remove  the  coarse  particles.  The  starch  rapidly  settles 
to  the  bottom  and  can  be  washed  by  repeated  decantation.  Allow 
the  compact  mass  of  starch  to  drain  thoroughly  and  spread  it  out 
on  a  watch  glass  to  dry  in  the  air.  If  so  desired  this  preparation  may 
be  used  in  the  experiments  which  follow. 

2.  Microscopical  Examination. — Examine  microscopically  the 
granules  of  the  various  starches  submitted  and  compare  them  with 
those  shown  in  Figs.  7-17,  page  44.  The  suspension  of  the  granules 
in  a  drop  of  water  will  facilitate  the  microscopical  examination. 

3.  Solubility. — Try  the  solubility  of  one  form  of  starch  in  each 
of  the  ordinary  solvents   (see  page  22).     If  uncertain  regarding  the 


44 


PHYSIOLOGICAL    CH F.MISTRY. 


Fig.  7. — Potato. 


Fig.  S.— Be.an. 


Fig.  o. — .Arrowroot. 


( 

^ 

1p 

•3 

% 

Fig.  ic. — Rye. 


Fig.  II. — B.'\RLEY. 


Vic.  12.     O.vr. 


!(t' 

l^.- 

^#>!F 

Fig.  13. — Buckwheat. 


Fig.  14.— .M.aize. 


Fig.  1^. — Rice. 


Fig.  16.     I'l.A.  I"..  17      Wheat. 

Sr.VRfH   Granui.ks    irom   Various   Soircks.     (Lrjhmui   mul   l>nini.) 


CARBOHYDRATES.  •  45 

solubility  in  any  reagent,  filter  and  test  the  filtrate  with  iodine  solution 
as  given  under  5  below.  The  production  of  a  blue  color  would  indi- 
cate that  the  starch  had  been  dissolved  by  the  solvent. 

4.  Iodine  Test. — Place  a  few  granules  of  starch  in  one  of  the 
depressions  of  a  porcelain  test-tablet  and  treat  with  a  drop  of  a  dilute 
solution  of  iodine  in  potassium  iodide.  The  granules  are  colored 
blue  due  to  the  formation  of  so-called  iodide  of  starch.  The  cellulose 
of  the  granule  is  not  stained  as  may  be  seen  by  examining  micro- 
scopically. 

5.  Iodine  Test  on  Starch  Paste. ^ — Repeat  the  iodine  test  using 
the  starch  paste.  Place  2-3  c.c.  of  starch  paste^  in  a  test-tube,  add 
a  drop  of  the  dilute  iodine  solution  and  observe  the  production  of  a 
blue  color.  Heat  the  tube  and  note  the  disappearance  of  the  color. 
It  reappears  on  cooling. 

In  similar  tests  note  the  influence  of  alcohol  and  of  alkali  upon 
the  so-called  iodide  of  starch. 

The  composition  of  the  iodide  of  starch  is  not  definitely  known. 

6.  Fehling's  Test. — On  starch  paste  (see  page  27). 

7.  Hydrolysis  of  Starch. — Place  about  25  c.c.  of  starch  paste 
in  a  small  beaker,  add  10  drops  of  concentrated  HCl,  and  boil.  By 
means  of  a  small  pipette,  at  the  end  of  each  minute,  remove  a  drop  of 
the  solution  to  the  test-tablet  and  make  the  regular  iodine  test.  As  the 
testing  proceeds  the  blue  color  should  gradually  fade  and  finally  dis- 
appear. At  this  point,  after  cooling  and  neutralizing  with  solid  KOH, 
Fehling's  test  (see  page  27)  should  give  a  positive  result  due  to  the 
formation  of  a  reducing  sugar  from  the  starch.  Make  the  phenyl- 
hydrazine  test  upon  some  of  the  hydrolyzed  starch.  What  sugar  has 
been  formed? 

8.  Influence  of  Tannic  Acid. — Add  an  excess  of  tannic  acid 
solution  to  a  small  amount  of  starch  paste  in  a  test-tube.  The  liquid 
will  become  strongly  opaque  and  ordinarily  a  yellowish- white  precipi- 
tate is  produced.  Compare  this  result  with  the  result  of  the  similar 
experiment  on  dextrin  (page  48). 

9.  Diffusibility  of  Starch  Paste. — Test  the  diffusibility  of  starch 
paste  through  animal  membrane  or  parchment  paper,  making  a  dia- 
lyzer  like  one  of  the  models  shown  in  Fig.  i,  page  25. 

^  Preparatian  of  Starch  Paste.  — Grind  2  grams  of  starch  powder  in  a  mortar  with  a  small 
amount  of  cold  water.  Bring  200  c.c.  of  water  to  the  boiling-point  and  add  the  starch 
mixture  from  the  mortar  with  continuous  stirring.  Bring  again  to  the  boiUng-point  and 
allow  it  to  cool.  This  makes  an  approximate  i  per  cent  starch  paste  which  is  a  verj- 
satisfactory  strength  for  general  use. 

^  For  this  particular  test  a  starch  paste  of  very  satisfactory  strength  may  be  made  by 
mixing  i  c.c.  of  a  i  per  cent  starch  paste  with  100  c.c.  of  water. 


46  PHYSIOLOGICAL   CHEMISTRY. 

INULIN,    (C,H,,0,),. 

Inulin  is  a  polysaccharide  which  may  be  obtained  as  a  white,  odor- 
less, tasteless  powder  from  the  tubers  of  the  artichoke,  elecampane,  or 
dahlia.  It  has  also  been  prepared  from  the  roots  of  chicory,  dandelion, 
and  Inirdock.  It  is  very  slightly  soluble  in  cold  water  and  quite  easily 
soluble  in  hot  water.  In  cold  alcohol  of  60  per  cent  or  over  it  is  prac- 
tically insoluble.  Inulin  gives  a  negative  reaction  with  iodine  solu- 
tion. The  "yellow''  color  reaction  with  iodine  mentioned  in  many 
books  is  doubtless  merely  the  normal  color  of  the  iodine  solution.  It  is 
very  dithcult  to  prepare  inulin  which  does  not  reduce  Fehling's  solu- 
tion slightly.  This  reducing  power  may  be  due  to  an  impurity.  Prac- 
tically all  commercial  preparations  of  inulin  possess  considerable  re- 
ducing power. 

Inulin  is  Icevo-rotatory  and  upon  hydrolysis  by  acids  or  by  the 
enzyme  inulase  it  yields  the  monosaccharide  laevulose  which  readily 
reduces  Fehling's  solution.  The  ordinary  amylolytic  enzymes  occur- 
ring in  the  animal  body  do  not  digest  inulin. 

Experiments  on  Inulin. 

r.  Solubility. — Try  the  solubility  of  inulin  powder  in  each  of 
the  ordinary  solvents.  If  uncertain  regarding  the  solubility  in  any 
reagent,  filter  and  neutralize  the  filtrate  if  it  is  alkaline  in  reaction. 
Add  a  drop  of  concentrated  hydrochloric  acid  to  the  filtrate  and  boil 
it  for  one  minute.  Render  the  solution  neutral  or  slightly  alkaline 
with  solid  potassium  hydroxide  and  try  Fehling's  test.  What  is  the 
significance  of  a  positive  Fehling's  test  in  this  connection  ? 

2.  Iodine  Test. — (a)  Place  2-3  c.c.  of  the  inulin  solution  in  a 
test-tube  and  add  a  drop  of  dilute  iodine  solution.  What  do  you 
observe  ? 

(b)  Place  a  small  amount  of  inulin  powder  in  one  of  the  depressions 
of  a  te.st-tablct  and  add  a  drop  of  dilute  iodine  solution.  Is  the  effect 
any  different  from  that  observed  above? 

3.  Molisch's  Reaction. — Repeat  this  test  according  to  directions 
gi\en  under  Dextrose,  2,  i)agc  22. 

4.  Fehling's  Test.  Make  this  test  on  the  inulin  solution  accord- 
ing to  the  instructions  given  under  Dextrose,  page  27.  Is  there  any 
reduction  ?' 

5.  Hydrolysis  of  Inulin.  Place  5  c.c.  of  inuh'n  solution  in  a 
test-tube,  add  a  dn^p  of  concentrated  hydrochloric  acid  and  boil  it 
for  one  minute.     Now  cool  the  solution,  neutralize  it  with  concentrated 

'  See  the  (lisfussion  f)f  (lie  i)rf)[)(;rtics  rif  inulin,  .ihove. 


CARBOHYDRATES.  47 

KOH  and  test  the  reducing  action  of  i  c.c.  of  the  solution  upon  i  c.c. 
of  diluted  (i  :4)  FehHng's  solution.     Explain  the  result.^ 

GLYCOGEN,    (C„H,,0,),. 

(For  discussion  and  experiments  see  Muscular  Tissue,  Chapter 
XV.) 

LICHENIN,    (C^Hj^OJ,. 

Lichenin  may  be  obtained  from  Cetraria  islandica  (Iceland  moss). 
It  forms  a  difficultly  soluble  jelly  in  cold  water  and  an  opalescent 
solution  in  hot  water.  It  is  optically  inactive  and  gives  no  color  with 
iodine.  Upon  hydrolysis  with  dilute  mineral  acids  lichenin  yields  dex- 
trins  and  dextrose.  It  is  said  to  be  most  nearly  related  chemically  to 
starch.  Saliva,  pancreatic  juice,  malt  diastase  and  gastric  juice  have 
no  noticeable  action  on  lichenin. 

DEXTRIN,    (CgHjoOg)^. 

The  dextrins  are  the  bodies  formed  midway  in  the  stages  of  the 
hydrolysis  of  starch  by  weak  acids  or  an  enzyme.  They  are  amor- 
phous bodies  which  are  easily  soluble  in  water,  acids,  and  alkalis,  but 
are  insoluble  in  alcohol  or  ether.  Dextrins  are  dextro-rotatory  and  are 
not  fermentable  by  yeast. 

The  dextrins  may  be  hydrolyzed  by  dilute  acids  to  form  dextrose. 
With  iodine  one  form  of  dextrin  (erythro-dextrin)  gives  a  red  color. 
Their  power  to  reduce  Fehling's  solution  is  questioned. 

Experiments  on  Dextrin. 

1.  Solubility. — Test  the  solubility  of  pulverized  dextrin  in  the 
ordinary  solvents  (see  page  22). 

2.  Iodine  Test. — Place  a  drop  of  dextrin  solution  in  one  of  the 
depressions  of  the  test-tablet  and  add  a  drop  of  a  dilute  solution  of 
iodine  in  potassium  iodide.  A  red  color  results  due  to  the  formation 
of  the  red  iodide  of  dextrin.  If  the  reaction  is  not  sufficiently  pro- 
nounced make  a  stronger  solution  from  pulverized  dextrin  and  repeat 
the  test.     The  solution  should  be  slightly  acid  to  secure  the  best  results. 

Make  proper  tests  to  show  that  the  red  iodide  of  dextrin  is  influenced 
by  heat,  alkali,  and  alcohol  in  a  similar  manner  to  the  blue  iodide  of 
starch  (see  page  45). 

'  If  the  inulin  solution  gave  a  positive  Fehling  test  in  the  last  experiment  it  will  be 
necessary  to  check  the  hydrolysis  experiment  as  follows:  To  5  c.c.  of  the  inulin  solution  in  a 
test-tube  add  one  drop  of  concentrated  hydrochloric  acid,  neutraUze  with  concentrated 
KOH  solution  and  test  the  reducing  action  of  i  c.c.  of  the  resulting  solution  upon  i  c.c. 
of  diluted  (i  :  4)  Fehling's  solution.  This  will  show  the  normal  reducing  power  of  the  inulin 
solution.  In  ca  e  the  inuUn  was  hydrolyzed,  the  Fehling's  test  in  the  hydrolysis  experiment 
should  show  a  more  pronounced  reduction  than  that  observed  in  the  check  experiment. 


48  PHYSIOLOGICAL    CHEMISTRY. 

3.  Fehling's  Test. — See  if  the  dextrin  solution  will  reduce  Feh- 
ling's  solution. 

4.  Hydrolysis  of  Dextrin. — Take  25  c.c.  of  dextrin  solution  in 
a  small  beaker,  add  5  drops  of  dilute  hydrochloric  acid,  and  boil.  Bv 
means  of  a  small  pipette,  at  the  end  of  each  minute,  remove  a  drop  of 
the  solution  to  one  of  the  depressions  of  the  test-tablet  and  make  the 
iodine  test.  The  power  of  the  solution  to  produce  a  color  with  iodine 
should  rapidly  disappear.  When  a  negative  reaction  is  obtained  cool 
the  solution  and  neutralize  it  with  concentrated  potassium  hydroxide. 
Try  Fehling's  test  (see  page  27).  This  reaction  is  now  strongly  posi- 
tive, due  to  the  formation  of  a  reducing  sugar.  Determine  the  nature 
of  the  sugar  by  means  of  the  phenylhydrazine  test  (see  pages  23  and  24). 

5.  Influence  of  Tannic  Acid. — Add  an  excess  of  tannic  acid 
solution  to  a  small  amount  of  dextrin  solution  in  a  test-tube.  No 
precipitate  forms.  This  result  diflfers  from  the  result  of  the  similar 
experiment  upon  starch  (see  Starch,  8,  page  45). 

6.  Diffusibility  of  Dextrin. —  (See  Starch,  9,  page  45.) 

7.  Precipitation  by  Alcohol. — To  about  50  c.c.  of  95  per  cent 
alcohol  in  a  small  beaker  add  about  10  c.c.  of  a  concentrated  dextrin 
solution.  Dextrin  is  thrown  out  of  solution  as  a  gummy  white  ])rc- 
cipitate.  Compare  the  result  with  that  obtained  under  Dextrose, 
5,  page  45. 

CELLULOSE,   (CgH.oOj^. 

This  complex  polysaccharide  forms  a  large    portion    of   the   cell 

wall  of  plants.     It  is  extremely  insoluble  and  its  molecule  is  much 

more  complex  than  the  starch  molecule.     The  best  (juality  of  filter 

paper  and  the  ordinary  absorbent  cotton  are  good  types  of  cellulose. 

Experiments  on   Cellulosj;. 

1.  Solubility. — Test  the  solubility  of  cellulose  in  the  ordinary 
solvents  (see  page  22 j. 

2.  Iodine  Test. — Add  a  dro])  (jf  dilute  iodine  solution  to  a  few 
shreds  of  cotton  on  a  test-tablet.  Cellulose  dilTers  from  starch  and 
flextrin  in  giving  no  color  with  iodine. 

3.  Formation  of  Amyloid. '     Add   10  ex.  of  dilute  and  5  c.c. 

of   concentrated    JIjSO^    to   some   absorbent    (olton    in    a    test-tube. 

When  entirely  dissolved  fwilhoul  heating)  pour  one-half  of  the  solution 

into  another  test-tube,  cool  it  and  dilute  with  water.     .Amyloid  forms  as 

a  gummy  precipitate  and  gives  a  brown  or  blue  coh^ration  with  iodine. 

'This  bofly  derives  its  name  from  tiniylum  (starch)  and  is  not  lo  he  i  onfoundcd  willi 
amyloid,  the  f;ly((jprotein. 


CARBOHYDRATES. 


49 


After  allowing  the  second  portion  of  the  acid  solution  of  cotton 
to  stand  about  lo  minutes,  dilute  it  with  water  in  a  small  beaker  and 
boil  for  15-30  minutes.  Now  cool,  neutralize  with  solid  KOH  and 
test  with  Fehling's  solution.  Dextrose  has  been  formed  from  the 
cellulose  by  the  action  of  the  acid. 

4.  Schweitzer's  Solubility  Test. — Place  a  little  absorbent  cotton 
in  a  test-tube,  add  Schweitzer's  reagent,^  and  stir  the  cellulose  with 
a  glass  rod.  When  completely  dissolved  acidify  Jhe  solution  with 
acetic   acid.     An   amorphous    precipitate   of   cellulose    is    produced. 

5.  Cross  and  Bevan's  Solubility  Test.^ — Place  a  little  absorbent 
cotton  in  a  test-tube,  add  Cross  and  Bevan's  reagent,^  and  stir  the 
cellulose  with  a  glass  rod.  When  solution  is  complete  reprecipitate 
the  celluose  with  95  per  cent  alcohol. 

REVIEW  OF  CARBOHYDRATES. 

In  order  to  facilitate  the  student's  review  of  the  carbohydrates, 
the  preparation  of  a  chart  similar  to  the  appended  model  is  recom- 

MODEL  CHART  FOR  REVIEW  PURPOSES. 


Carbohydrate. 

"o 
m 

H 

■3 
0 

H 
0 

0 

H 

a 
2 

s 

"tuo 

.s 

is 
0 

'I 

c 

H 

■0 
u 

PQ 

0  0 

^  S 

2rt 

H 
< 

1 

23^ 

c    . 
.2-0 

u 

a, 

§ 

0 

c 
0 

3 
0 

1 

0 

CIS 
C 

E 

a 

E 

1  Dextrose. 







!  Lsevulose. 

1 



j  Maltose. 



— 

_ 

— 



Iso-maltose. 

t 

Lactose. 







— 







Sucrose, 

Starch. 

Inulin. 



Glycogen. 

Dextrin. 
Cellulose. 



" 

" 



mended.     The  signs  +  and  —  may  be  conveniently  used  to  indicate 
positive    and    negative    reaction.     Only    those    carbohydrates    which 

*  Schweitzer's  reagent  is  made  by  adding  potassium  hydroxide  to  a  solution  of  cupric 
sulphate  which  contains  some  ammonium  chloride.  A  precipitate  of  cupric  hydroxide 
forms  and  this  is  filtered  off,  washed,  and  3  grams  of  the  moist  cupric  hydroxide  brought 
into  solution  in  a  liter  of  20  per  cent  ammonium  hydroxide. 

^  Cross  and  Bevan:  Chemical  News,  63,  p.  66. 

^  Cross  and  Bevan's  reagent  may  be  prepared  by  combining  two  parts  of  concen- 
trated hydrochloric  acid  and  one  part  of  zinc  chloride,  by  weight. 

4 


so 


PHYSIOLOGICAL    CHEMISTRY. 


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CARBOHYDRATES. 


51 


are  of  greatest  importance  from  the  standpoint  of  physiological  chemis- 
try have  been  included  in  the  chart. 

"Unknown"  Solutions  of  Carbohydrates. 

At  this  point  the  student  will  be  given  several  "unknown"  solu- 
tions, each  solution  containing  one  or  more  of  the  carbohydrates 
studied.  He  will  be  required  to  detect,  by  means  of  the  tests  on  the 
preceding  pages,  each  carbohydrate  constituent  of  the  several  "un- 
known" solutions  and  hand  in,  to  the  instructor,  a  written  report 
of  his  findings,  on  slips  furnished  by  the  laboratory. 

The  scheme  given  on  page  50  may  be  of  use  in  this  connection. 


CHAPTER  HI. 
SALIVARY  DIGESTION. 

The  saliva  is  secreted  by  three  pairs  of  glands,  the  submaxillary, 
sublingual,  and  parotid,  reinforced  by  numerous  small  glands  called 
buccal  glands.  The  saliva  secreted  by  each  pair  of  glands  possesses 
certain  definite  characteristics  peculiar  to  itself.  For  instance,  in 
man  the  parotid  glands  ordinarily  secrete  a  thin,  watery  fluid,  the 
submaxillary  glands  secrete  a  somewhat  thicker  fluid  containing 
mucin,  while  the  product  of  the  sublingual  glands  has  a  more  muci- 
laginous character.  The  saliva  as  collected  from  the  mouth  is  the 
combined  product  of  all  the  glands  mentioned. 

The  saliva  may  be  induced  to  flow  by  many  forms  of  stimuli,  such 
as  chemical,  mechanical,  electrical,  thermal,  and  psychical,  the  nature 
and  amount  of  the  secretion  depending,  to  a  limited  degree,  upon 
the  particular  class  of  stimuli  employed  as  well  as  upon  the  character 
oi  the  individual  stimulus.  For  example,  in  experiments  upon  dogs 
it  has  been  found  that  the  mechanical  stimulus  afforded  by  dropping 
several  pebbles  into  the  animal's  mouth  caused  the  flow  of  but  one 
or  two  drops  of  saliva,  whereas  the  mechanical  stimulus  afforded 
by  sand  thrown  into  the  mouth  induced  a  copius  flow  of  a  thin  watery 
tluid.  Again,  when  ice-water  or  snow  was  placed  in  the  animal's 
mouth  no  sali\u  was  seen,  while  an  acid  or  anything  possessing  a 
bitter  taste,  which  the  dog  wished  to  reject,  caused  a  free  flow  of  the 
thin  saliva.  On  the  other  hand,  when  articles  of  food  were  placed  in 
the  dog's  mouth  the  animal  secreted  a  thicker  saliva  having  a  higher 
mucin  content  a  fluid  which  would  lubricate  the  food  and  assist  in 
the  passage  of  the  bolus  through  the  (esophagus.  It  was  further 
ffumd  that  by  simply  drawing  the  attention  of  the  animal  to  any  of 
the  substances  named  al)ove,  results  were  obtained  similar  to  those 
secured  when  the  substances  were  actually  placed  in  the  animal's 
mouth.  For  example,  when  a  pretense  was  made  of  throwing  sand 
into  the  dog's  mouth,  a  watery  saliva  was  secreted,  whereas  food  under 
the  same  conditions  exciled  a  thicker  and  more  slimy  secretion.  The 
exhibiti(m  of  dry  food,  in  which  the  dog  had  no  particular  interest 
(dry  brcadj  caused  the  secretion  o{  a  large  amount  of  watery  saliva, 
while  the  presentation  of  moist  food,  which  was  eagerly  desired  by  the 

.S2 


SALIVARY   DIGESTION.  53 

animal,  called  forth  a  much  smaller  secretion,  slimy  in  character. 
These  experiments  show  it  to  be  rather  difficult  to  differentiate  between 
the  influence  of  physiological  and  psychical  stimuli. 

The  amount  of  saliva  secreted  by  an  adult  in  24  hours  has  been 
variously  placed,  as  the  result  of  experiment  and  observation,  be- 
tween 1000  and  1500  c.c,  the  exact  amount  depending,  among  other 
conditions,  upon  the  character  of  the  food. 

The  saliva  ordinarily  has  a  weak,  alkaline  reaction  to  litmus,  but 
becomes  acid,  in  some  instances,  2-3  hours  after  a  meal  or  during 
fasting.  The  alkalinity  is  due  principally  to  di-sodium  hydrogen 
phosphate  (NajHPO^)  and  its  average  alkalinity  may  be  said  to  be 
equivalent  to  0.08-0.1  per  cent  sodium  carbonate.  The  saliva  is 
the  most  dilute  of  all  the  digestive  secretions,  having  an  average  spe- 
cific gravity  of  1.005  ^^^  containing  only  0.5  per  cent  of  solid  matter. 
Among  the  solids  are  found  albumin,  globulin,  mucin,  urea,  the  en- 
zymes salivary  amylase  (ptyalin)  and  maltase,  phosphates,  and  other 
inorganic  constitutents.  Potassium  thiocyanate,  KSCN,  is  also 
generally  present  in  the  saliva.  It  has  been  claimed  that  this  sub- 
stance is  present  in  greatest  amount  in  the  saliva  of  habitual  smokers. 
The  significance  of  thiocyanate  in  the  saliva  is  not  known;  it  probably 
comes  from  the  ingested  thiocyanates  and  from  the  breaking  down  of 
protein  material. 

The  so-called  tartar  formation  on  the  teeth  is  composed  almost 
entirely  of  calcium  phosphate  with  some  calcium  carbonate,  mucin, 
epithelial  cells,  and  organic  debris  derived  from  the  food.  The  cal- 
cium salts  are  held  in  solution  as  acid  salts,  and  are  probably  pre- 
cipitated by  the  ammonia  of  the  breath.  The  various  organic  sub- 
stances just  mentioned  are  carried  down  in  the  precipitation  of  the 
calcium  salts. 

The  principal  enzyme  of  the  saliva  is  known  as  salivary  amylase 
or  ptyalin.  This  is  an  amylolytic  enzyme  (see  p.  3),  so  called  because 
it  possesses  the  property  of  transforming  complex  carbohydrates 
such  as  starch  and  dextrin  into  simpler  bodies.  The  action  of  salivary 
amylase  is  one  of  hydrolysis  and  through  this  action  a  series  of  simpler 
l)odies  are  formed  from  the  complex  starch.  The  first  product  of 
the  action  of  the  ptyalin  of  the  saliva  upon  starch  paste  is  soluble  starch 
(amidulin)  and  its  formation  is  indicated  by  the  disappearance  of  the 
opalescence  of  the  starch  solution.  This  body  resembles  true  starch 
in  giving  a  blue  color  with  iodine.  Next  follows  the  formation,  in 
succession,  of  a  series  of  dextrins,  called  erythro-dextrin,  a-achroo- 
dextrin,    ^-achroo-dextrin,    and    y-achroo-dextrin,    the    erythro-dextrin 


54  PHYSIOLOGICAL    CHEMISTRY. 

being  formed  directly  from  soluble  starch  and  later  being  itself  trans- 
formed into  oc-achroo-dextrin  from  which  in  turn  are  produced  ^-achroo- 
dextrin  and  j-ackroo-dextrin.  Accompanying  each  dextrin  a  small 
amount  of  iso-maltose  is  formed,  the  quantity  of  iso-maltose  growing 
gradually  larger  as  the  process  of  transformation  progresses.  (Erythro- 
dextrin  gives  a  red  color  with  iodine,  the  other  dcxtrins  give  no  color.) 
The  next  stage  is  the  transformation  of  the  y-achroo-dexirin  into  iso- 
maltose  and  subsequently  the  transformation  of  the  iso-maltose  into 
maltose,  the  latter  being  the  principal  'end-product  of  the  salivary 
digestion  of  starch.  At  this  point  a  small  amount  of  dextrose  is  formed 
from  the  maltose,  through  the  action  of  the  enzyme  maltase. 

Salivary  amylase  acts  in  alkaline,  neutral,  or  combined  acid  solu- 
tions. It  will  act  in  the  presence  of  relatively  strong  combined  HCl 
(see  page  409),  whereas  a  trace  (0.003  pcr  cent  to  0.006  per  cent)  of 
ordinary  free  hydrochloric  acid  will  not  only  prevent  the  action  but 
will  destroy  the  enzyme.  By  sufficiently  increasing  the  alkalinity  of 
the  saliva  to  litmus,  the  action  of  the  salivary  amylase  is  inhibited.  Jt 
has  recently  been  shown  by  Cannon  that  sali\ar}^  digestion  may  pro- 
ceed for  a  considerable  period  after  the  food  reaches  the  stomach, 
owing  to  the  slowness  with  which  the  contents  arc  thoroughly  mixed 
with  the  acid  gastric  juice  and  the  conseciucnt  tardy  destruction  of  the 
enzyme.  Food  in  the  pyloric  end  of  the  stomach  is  soon  mixed  with 
the  gastric  secretion,  but  food  in  the  cardiac  end  is  not  mixed  with  the 
acid  gastric  juice  for  a  considerable  period  of  time,  and  in  this  region 
during  that  time  sali\ary  digestion  may  ])r()cee(l  undisturbed. 

Maltase,  sometimes  called  glucase,  is  the  second  enzyme  of  the 
saliva.  It  is  an  amylolytic  enzyme  whose  principal  function  is  the 
splitting  of  maltose  into  dextrose.  Besides  occurring  in  the  saliva  it 
is  also  present  in  the  pancreatic  and  intestinal  juices.  For  experi- 
mental purposes  the  enzyme  is  ordinarily  prepared  from  corn.  'I'he 
j>rincijjlcs  (A  the  "reversibility"  of  enzyme  action  were  first  demon- 
strated in  cfjnnection  with  maltase  by  Croft  Hill. 

Microscopical  examination  of  the  saliva  reveals  salivary  corpus- 
cles, bacteria,  food  ddbris,  epithelial  cells,  mucus,  and  fungi.  In  cer- 
tain pathological  conditions  of  the  mouth,  pus  cells,  and  blood  corpus- 
cles may  be  fountl  in  I  he  saliva. 

EXPERIMEN'IS    ON    SaLIVA. 

A  satisfactory  met  hod  of  ohlaining  the  saliva  necessary  for  the 
experiments  which  follow  is  to  chew  a  small  piece  of  pure  j)aranin 
wax,  thus  stimulating  the  flow  of  the  secretion,  which  may  be  collected 


SALIVARY    DIGESTION.  55 

in  a  smalt  beaker.     Filtered  saliva  is  to  be  used  in  every  experiment 
except  for  the  microscopical  examination. 

1.  Microscopical  Examination. — Examine  a  drop  of  unfiltered 
saliva  microscopically  and  compare  with  Fig.  i8  below. 

2.  Reaction. — Test  the  reaction  to  litmus. 

3.  Specific   Gravity. — Partially  fill   a   urinometer   cylinder   with 
saliva,  introduce  the  urinometer,  and  observe  the  reading. 

4.  Test  for  Mucin. — To  a  small  amount  of  saliva  in  a  test-tube 
add  1-2  drops  of  dilute  acetic  acid.     Mucin  is  precipitated. 


%t'^^' 


Fig.  18. — Microscopical  Constituents  of  Saliva. 
a,  Epithelial  cells;  b,  salivary  corpuscles;  c,  fat  drops;  d,  leucocytes;  e,  /  and  g,  bacteria; 

h,  i  and  k,  fission-fungi. 

5.  Biuret  Test.^ — Render  a  little  saliva  alkaline  with  an  ec|ual 
volume  of  KOH  and  add  a  few  drops  of  a  very  dilute  (2-5  drops  in  a 
test-tube  of  water)  cupric  sulphate  solution.  The  formation  of  a 
purplish- violet  color  is  due  to  mucin. 

6.  Millon^s  Reaction.^ — Add  a  few  drops  of  Millon's  reagent  to 
a  little  saliva.  x\  light  yellow  precipitate  formed  by  the  mucin  gradu- 
ally turns  red  upon  being  gently  heated. 

7.  Preparation  of  Mucin. — Pour  25  c.c.  of  saliva  into  100  c.c. 
of  95  per  cent  alcohol,  stirring  constantly.  Cover  the  vessel  and  allow 
the  precipitate  to  stand  at  least  12  hours.  Pour  off  the  supernatant 
liquid,  collect  the  precipitate  on  a  filter  and  wash  it,  in  turn,  with 
alcohol  and  ether.  Finally  dry  the  precipitate,  remove  it  from  the 
paper  and  make  the  following  tests  on  the  mucin:  '  (a)  Test  its  solu- 
bility in  the  ordinary  solvents  (see  page  22);  {h)  Millon's  reaction; 
(c)  dissolve  a  small  amount  in  KOH,  and  try  the  biuret  test  on  the 
solution;  {d)  boil  the  remainder,  with  10-25  c.c.  of  water  to  which  5  c.c. 
of  dilute  HCl  has  been  added,  until  the  solution  becomes  brownish. 
Cool,  render  alkaline  with  solid  KOH,  and  test  by  Fehling's  solution. 
A  reduction  should  take  place.     Mucin  is  what  is  known  as  a  conju- 

'  The  significance  of  this  reaction  is  pointed  out  on  page  go. 
-  The_^significance  of  this  reaction  is  pointed  out  on  page  88. 


50  PHYSIOLOGICAL    CHEMISTRY. 

gated  protein  or  glycoprotein  (see  p.  85)  and  upon  boiling  with  the 
acid  the  carbohydrate  group  in  the  molecule  has  been  split  off  from  the 
protein  portion  and  its  presence  is  indicated  by  the  reduction  of 
Fehling's  solution. 

8.  Inorganic  Matter. — Test  for  chlorides,  phosphates,  sulphates, 
and  calcium.  For  chlorides,  acidify  with  HNO3  ^^^  ^^^  AgNOg. 
For  phosphates,  acidify  with  HNO3,  heat  and  add  molybdic  solution.^ 
For  sulphates,  acidify  with  HCl  and  add  BaClj  and  warm.  For  cal- 
cium, acidify  with  acetic  acid.  CH3COOH,  and  add  ammonium  oxa- 
late, (NHJ2C,0,. 

9.  Viscosity  Test. — Place  filter  papers  in  two  funnels,  and  to 
each  add  an  equal  quantity  of  starch  paste  (5  c.c).  Add  a  few  drops 
of  saliva  to  one  lot  of  paste  and  an  equivalent  amount  of  water  to  the 
other.  Note  the  progress  of  filtration  in  each  case.  Why  does  one 
solution  filter  more  rapidly  than  the  other? 

10.  Test  for  Nitrites. — Add  1-2  drops  of  dilute  HjSO^  to  a  little 
saliva  and  thoroughly  stir.  Now  add  a  few  drops  of  a  potassium 
iodide  solution  and  some  starch  paste.  Nitrous  acid  is  formed  which 
liberates  iodine,  causing  the  formation  of  the  blue  iodide  of  starch. 

11.  Thiocyanate  Tests. —  (a)  Ferric  Chloride  Test. — To  a  little 
saliva  in  a  small  porcelain  crucible,  or  dish,  add  a  few  drops  of  dilute 
ferric  chloride  and  acidify  slightly  with  HCl.  Red  ferric  thiocyanate 
forms.  To  show  that  the  red  coloration  is  not  due  to  iron  phosphate 
add  a  drop  of  HgClj  when  colorless  mercuric  thiocyanate  forms. 

(b)  Solera's  Reaction. — This  test  depends  upon  the  liberation  of 
iodine  through  the  action  of  thiocyanate  upon  iodic  acid.  Moisten 
a  .strip  of  starch  paste-iodic  acid  test  paper^  with  a  little  saliva.  If 
thiocyanate  be  present  the  test  paper  will  assume  a  blue  color,  due  to 
the  liberation  of  iodine  and  the  subsequent  formation  of  the  so-called 
iodide  of  starch. 

12.  Digestion  of  Starch  Paste. — To  25  c.c.  of  starch  paste  in 
a  small  Ijcaker,  add  5  drops  of  saliva  and  stir  thoroughly.  At  intervals 
of  a  minute  remove  a  drop  of  the  solution  to  one  of  the  dei)ressions  in 
a  test-tablet  and  test  by  the  iodine  test.  If  the  blue  color  with  iodine 
still  forms  after  5  minutes,  add  another  5  drops  of  saliva.     The  opal- 

'  Molybdic  solution  is  prepared  as  follows,  tlic  i>arls  Ijcin^  by  weight: 
I  part  inolybdic  acid. 

4  |)arts  ammonium  hydroxide  (sjj.  gr.  0.96). 
15  parts  nitric  acid  (  j).  gr.  i  .2). 
-'  This  test  paper  is  preparer!  as  foll(jws:  Saturate  a  good  quality  of  filter  paper  with 
0.5  per  cent  starch  jjasle  to  which  has  l)een  added  sufTicient  iodic  acid  to  make  a  i  per  cent 
s<^)!ution  of  iodic  acid  and  allow  the  paper  to  dry  in  the  air.     Cut  it  in  strips  of  suitable 
size  anfl  preserve  for  use. 


SALIVARY   DIGESTION.  57 

escence  of  the  starch  solution  should  soon  disappear,  indicating  the 
formation  of  soluble  starch  which  gives  a  blue  color  with  iodine.  This 
body  should  soon  be  transformed  into  ery  thro -dextrin  which  gives  a  red 
color  with  iodine,  and  this  in  turn  should  pass  into  achroo-dextrin  which 
gives  no  color  with  iodine.  This  is  called  the  achromic  point.  When 
this  point  is  reached  test  by  Fehling's  test  to  show  the  production  of  a 
reducing  body.  A  positive  Fehling's  test  may  be  obtained  while  the 
solution  still  reacts  red  with  iodine  inasmuch  as  some  iso-maltose  is 
formed  from  the  soluble  starch  coincidently  with  the  formation  of  the 
erythro-dextrin.  How  long  did  it  take  for  a  complete  transformation 
of  the  starch  ? 

13.  Digestion  of  Dry  Starch. — In  a  test-tube  shake  up  a  small 
amount  of  dry  starch  with  a  little  water.  Add  a  few  drops  of  saliva, 
mix  well,  and  allow  to  stand.  After  10-20  minutes  filter  and  test  the 
filtrate  by  Fehling's  test.     What  is  the  result  and  why  ? 

14.  Digestion  of  Inulin. — To  5  c.c.  of  inulin  solution  in  a  test- 
tube  add  10  drops  of  saliva  and  place  the  tube  in  the  water-bath  at  40° 
C.  After  one-half  hour  test  the  solution  by  Fehling's  test.^  Is  any 
reducing  substance  present?  What  do  you  conclude  regarding  the 
salivary  digestion  of  inulin? 

15.  Influence  of  Temperature. — In  each  of  four  tubes  place 
about  5  c.c.  of  starch  paste.  Immerse  one  tube  in  cold  water  from 
the  faucet,  keep  a  second  at  room  temperature,  and  place  a  third  on  the 
water-bath  at  40°  C.  Now  add  to  the  contents  of  each  of  these  three 
tubes  two  drops  of  saliva  and  shake  well;  to  the  contents  of  the  fourth 
tube  add  two  drops  of  boiled  saliva.  Test  frequently  by  the  iodine 
test,  using  the  test-tablet,  and  note  in  which  tube  the  most  rapid  diges- 
tion occurs.     Explain  the  results. 

16.  Influence  of  Dilution. — Take  a  series  of  six  test-tubes  each 
containing  9  c.c.  of  water.  Add  i  c.c.  of  saliva  to  tube  i  and  shake 
thoroughly.  Remove  i  c.c.  of  the  solution  from  tube  i  to  tube  2  and 
after  mixing  thoroughly  remove  i  c.c.  from  tube  2  to  tube  3.  Continue 
in  this  manner  until  you  have  6  saliva  solutions  of  gradually  decreasing 
strength.  Now  add  starch  paste  in  equal  amounts  to  each  tube,  mix 
very  thoroughly,  and  place  on  the  water-bath  at  40°  C.  After  10-20 
minutes  test  by  both  the  iodine  and  Fehling's  tests.  In  how  great 
dilution  does  your  saliva  act? 

17.  Influence  of  Acids  and  Alkalis. — (a)  Influence  of  Free 
Acid. — Prepare  a  series  of  six  tubes  in  each  of  which  is  placed  4  c.c. 

■  If  the  inulin  solution  gives  a  reduction  before  being  acted  upon  by  the  saliva  it  will  be 
necessary  to  determine  the  extent  of  the  original  reduction  by  means  of  a  "check"  test 
(see  page  47). 


58  PHYSIOLOGICAL    CHEMISTRY. 

of  one  of  the  following  strengths  oifree  HCl:  0.2  per  cent,  o.i  per  cent, 
0.05  per  cent",  0.025  per  cent,  0.0125  per  cent  and  0.006  per  cent.  Now 
add  2  c.c.  of  starch  paste  to  each  tube  and  shake  them  thoroughly. 
Complete  the  solutions  by  adding  2  c.c.  of  saliva  to  each  and  repeat 
the  shaking.  The  total  acidity  of  this  series  would  be  as  follows:  o.i 
per  cent,  0.05  per  cent.  0.025  per  cent,  0.0125  per  cent,  0.006  per  cent 
and  0.003  P^r  cent.  Place  these  tubes  on  the  water-bath  at  40°  C. 
for  10-20  minutes.  Divide  the  contents  of  each  tube  into  two 
parts,  testing  one  part  by  the  iodine  test  and  testing  the  other,  after 
neutralization,  by  Fehling's  test.     What  do  you  find? 

{b)  Influence  of  Combined  Acid. — Repeat  the  first  three  experiments 
of  the  above  series  using  combined  hydrochloric  acid  (see  page  409) 
instead  of  the  free  acid.  How  does  the  action  of  the  combined  acid 
differ  from  that  of  t\iQ  free  acid? 

(c)  Influence  of  Alkali. — Repeat  the  first  four  experiments  under 
(a)  replacing  the  HCl  by  2  per  cent,  r  per  cent,  0.5  per  cent  and  0.25 
per  cent  Na2C03.  Neutralize  the  alkalinity  before  trying  the  iodine 
test  (see  Starch,  5,  page  45). 

(d)  Nature  of  the  Action  of  Acid  and  Alkali. — Place  2  c.c.  of  saliva 
and  2  c.c.  of  0.2  per  cent  HCl  in  a  test-tube  and  leave  for  15  minutes. 
Neutralize  the  solution,  add  4  c.c.  of  starch  paste  and  place  the  tube 
on  the  water-bath  at  40°  C.  In  10  minutes  test  by  the  iodine  and 
Fehling's  tests  and  explain  the  result.  Rei)cat  the  experiment,  replac- 
ing the  0.2  per  cent  HCl  by  2  per  cent  Na^COj.  What  do  you  deduce 
from  these  two  experiments? 

18.  Influence  of  Metallic  Salts,  etc.  In  each  of  a  series  of  tubes 
place  4  c.c.  of  starch  paste  and  1/2  c.c.  of  one  of  the  solutions  named 
below.  Shake  well,  add  r/2  c.c.  of  sali\a  to  each  tube,  thoroughly 
mix,  and  place  on  the  water-bath  at  40^^  C.  for  10-20  minutes.  Show 
the  progress  of  digestion  by  means  of  the  iodine  and  Fehling  tests. 
Use  the  following  chemicals:  Metallic  salts,  1  o  per  cent  plumbic  acetate, 
2  per  cent  cupric  sulphate,  5  per  cent  ferric  chloride,  8  per  cent  mercuric 
chloride;  Neutral  salts,  jo  per  cent  sodium  chloride,  jo  ])er  cent  mag- 
nesium sulphate,  3  per  cent  barium  chloride,  ro  per  cent  Rochelie  salt. 
Also  try  the  intluencc  of  2  |)er  cent  carb(jlic  acid,  ()5  per  cent  alcohol, 
and  ether  and  chloroform.      What  arc  your  conclusi(jns  ? 

19.  Excretion  of  Potassium  Iodide,  ingest  a  small  dose  of 
potassium  iodide  (0.2  gramj  contained  in  a  gelatin  capsule,  (|uickly 
rinse  out  the  mouth  with  water,  and  then  test  the  saliva  at  once  for 
iodine.  This  test  should  be  negative.  Make  additional  tests  for 
iodine  at  2-minute  intervals.     The  test  for  iodine  is  made  as  follows: 


■      SALIVARY    DIGESTION.  59 

Take  i  c.c.  of  NaNOj  and  i  ex.  of  dilute  H2S04^  in  a  test-tube,  add 
a  little  saliva  directly  from  the  mouth,  and  a  small  amount  of  starch 
paste.  If  convenient,  the  urine  may  also  be  tested.  The  formation 
of  a  blue  color  signifies  that  the  potassium  iodide  is  being  excreted 
through  the  salivary  glands.  Note  the  length  of  time  elapsing  between 
the  ingestion  of  the  potassium  iodide  and  the  appearance  of  the  first 
traces  of  the  substance  in  the  saliva.  The  chemical  reactions  taking 
place  in  this  experiment  are  indicated  in  the  following  equations: 

(a)  2NaN02  +  H2SO,  =  2HN02  +  Na2SO,. 

(b)  2KI  +  H2SO,  =  2HI  +  K2S04. 

(c)  2HN02  +  2HI  =  l2  +  2H20-;:-2NO. 

20.  Qualitative  Analysis  of  the  Products  of  Salivary  Diges- 
tion.— To  25  c.c.  of  the  products  of  salivary  digestion  (saved  from 
Experiment  12  or  furnished  by  the  instructor),  add  100  c.c.  of  95  per 
cent  alcohol.  Allow  to  stand  until  the  white  precipitate  has  settled. 
Filter,  evaporate  the  filtrate  to  dryness,  dissolve  the  residue  in  5-10 
c.c.  of  water  and  try  Fehling's  test  (p.  27)  and  the  phenylhydrazine 
reaction  (see  Dextrose,  3,  page  23).  On  the  dextrin  precipitate  try 
the  iodine  test  (page  45).  Also  hydrolyze  the  dextrin  as  given  under 
Dextrin,  4,  page  48. 

^  Instead  of  this  mixture  a  few  drops  of  HNO3  possessing  a  yellowish  or  brownish 
color  due  to  the  presence  of  HNO^  may  be  employed. 


CHAPTER  IV. 
PROTEINS:^     THEIR  DECOMPOSITION  AND  SYNTHESIS. 

The  proteins  are  a  class  of  substances,  which  in  the  light  of  our 
present  knowledge,  consist,  in  the  main,  of  combinations  of  a-amino- 
acids  or  their  deri\ati\es.  These  protein  substances  form  the  chief 
constituents  of  many  of  the  fluids  of  the  body,  constitute  the  organic 
l)asis  of  animal  tissue,  and  at  the  same  time  occupy  a  decidedly  pre- 
eminent position  among  our  organic  food-stuffs.  They  are  absolutely 
necessary  to  the  uses  of  the  animal  organism  for  the  continuance  of 
life  and  they  cannot  be  satisfactorily  replaced  in  the  diet  of  such  an 
organism  by  any  other  dietary  constituent  either  organic  or  inorganic. 
Such  an  organism  may  exist  without  protein  food  for  a  period  of  time, 
the  length  of  the  period  varying  according  to  the  specific  organism  and 
the  nature  of  the  substitution  offered  for  the  protein  portion  of  the  diet. 
Such  a  period  is,  however,  distinctly  one  of  existence  rather  than  one  of 
normal  life  and  one  which  is  consequently  not  accompanied  by  such 
a  full  and  free  exercise  of  the  various  functions  of  the  organism  as 
would  be  possible  upon  an  evenly  Ijalanced  ration,  i.  e.,  one  containing 
the  requisite  amount  of  protein  food.  These  protein  substances  are, 
furthermore,  essential  constituents  of  all  living  cells  and  therefore 
without  them  vegetable  life  as  well  as  animal  life  is  imi)ossible. 

The  proteins,  which  constitute  such  an  important  group  of  sub- 
stances, differ  from  the  carbohydrates  and  fats  very  decidedly  in  ele- 
mentary composition.  In  addition  to  containing  carbon,  hydrogen, 
and  oxygen,  which  are  present  in  fats  and  carbohydrates,  the  proteins 
invariably  contain  nitrogen  in  their  molecule  and  generally  sulphur 
also.  Proteins  ha\e  also  been  identified  which  contain  phosphorus, 
iron,  copper,  iodine,  manganese,  and  zinc.  The  ])ercentage  composition 
of  the  more  important  members  of  the  group  of  protein  substances 
would  fall  within  the  following  limits:  C==  50-55  per  cent,  H  =  6-7.3 
per  cent,  0  =  19-24  per  cent,  N=  15-19  per  cent,  S  =  0.3-2.5  per  cent, 
P  =  0.4-0.8  per  cent  when  present.     When  iron,  copper,  iodine,  manga- 

'  The  term  proteid  has  been  very  widely  used  by  English-speaking  scientists  to  signify 
the  class  of  substances  we  have  called  proteins. 

60 


PROTEINS.  6l 

nese,  or  zinc  are  present  in  the  protein  molecule  they  are  practically 
without  exception  present  only  in  traces} 

Of  all  the  various  elements  of  the  protein  molecule,  nitrogen  is  by 
far  the  most  important.  The  human  body  needs  nitrogen  for  the  con- 
tinuation of  life,  but  it  cannot  use  the  nitrogen  of  the  air  or  that  in  vari- 
ous other  combinations  as  we  find  it  in  nitrates,  nitrites,  etc.  How- 
ever, in  the  protein  molecule  the  nitrogen  is  present  in  a  form  which  is 
utilizable  by  the  body.  The  nitrogen  in  the  protein  molecule  occurs 
in  at  least /c^/r  different  forms  as  follows: 

I.  Monamino  acid  nitrogen. 

II.  Diamino  acid  nitrogen  or  basic  nitrogen. 

III.  Amide  nitrogen. 

IV.  A  guanidine  residue. 

The  actual  structure  of  the  protein  molecule  is  still  unknown,  and 
we  have  as  yet  no  means  by  which  its  molecular  weight  can  be  even 
approximately  established.  The  many  attempts  which  have  been 
made  to  determine  this  have  led  to  very  different  results,  some  of  which 
are  given  in  the  following  table: 

Serum  albumin    =     4572  — 5100  — 5135 

Egg  albumin        =  4900  —  6542 

Globin  =  15000— 16086 

Oxyhaemoglobin  =  14800— 15000— 16655  — 16730 

Of  these  figures,  those  given  for  oxyhaemoglobin  deserve  the  most 
consideration,  for  these  are  based  on  the  atomic  ratios  of  the  sul- 
phur and  iron  contained  in  this  substance.  The  simplest  formula 
that  can  be  calculated  from  analyses  of  oxyheemoglobin,  namely. 
C658Hii8i-'^207^2Fe02ioj  scrvcs  to  show  the  great  complexity  of  this 
substance.  The  following  formulas  which  have  been  proposed  for 
typical  protein  substances  may  serve  to  further  impress  the  fact  of 
the  great  size  of  the  protein  molecule: 

Egg  albumin        =  ^..z^^^^^^^s'^oOis 
Serum  albumin  =  C^soH^j^N^^gSgOj^o 

The  decomposition"  of  protein  substances  may  be  brought  about 
by  oxidation  or  hydrolysis,  but  inasmuch  as  the  hydrolytic  procedure 

*  Some  investigators  regard  these  elements  as  contaminations,  or  constituents  of  some 
non-protein  substance  combined  with  the  protein. 

^  The  terms  "degradation,"  "dissociation,"  and  "cleavage,"  are  often  used  in  tins 
connection. 


62  PHYSIOLOGICAL    CHEMISTRY. 

has  been  productix  e  of  the  more  satisfactory  results,  that  type  of  decom- 
position procedure  alone  is  used  at  present.  This  hydrolysis  of  the 
protein  molecule  may  be  accomplished  by  acids,  alkalis,  or  superheated 
steam,  and  in  digestion  by  the  action  of  the  proteolytic  enzymes.  The 
character  of  the  decomposition  products  varies  according  to  the  method 
utilized  in  tearing  the  molecule  apart.  Bearing  this  in  mind,  we  may 
say  that  the  decomposition  products  of  proteins  include  proteoses,  pep- 
tones, peptides,  carbon  dioxide,  ammonia,  hydrogen  sulphide,  and  amino 
acids.  These  amino  acids  constitute  a  long  list  of  important  substances 
which  contain  nuclei  belonging  either  to  the  aliphatic,  carbocyclic,  or 
heterocyclic  series.  The  list  includes  glycocoll,  alanine,  serine,  phenyl- 
alanine, tyrosine,  cystine,  tryptophane,  histidine,  valine,  arginine,  leucine, 
isoleucine,  lysine,  aspartic  acid,  glutamic  acid,  proline,  oxyproline,  and 
diaminotrihydroxydodecanoic  acid.  Of  these  amino  acids,  tyrosine 
and  phenylalanine  contain  carbocyclic  nuclei,  histidine,  proline,  and 
tryptophane  contain  heterocyclic  nuclei,  and  the  remaining  members 
of  the  list,  as  given,  contain  aliphatic  nuclei.  The  amino  acids  are 
preeminently  the  most  important  class  of  protein  decomposition 
products.  These  amino  acids  are  all  a-amino  acids,  and,  with  the 
exception  of  glycocoll,  are  all  optically  active.  Furthermore,  they  are 
amphoteric  substances  and  consequently  are  able  to  form  salts  with 
both  bases  and  acids.  These  properties  are  inherent  in  the  NH2  and 
COOH  groups  of  the  amino  acids. 

The  decomposition  products  of  protein  may  be  grouped  as  pri- 
mary and  secondary  decomposition  products.  By  primary  products 
are  meant  those  which  exist  as  radicals  within  the  protein  molecule 
and  which  are  liberated,  upon  cleavage  of  this  molecule,  with  their 
carbon  chains  intact  and  the  position  of  their  nitrogen  unaltered. 
The  secondary  products  are  those  which  result  from  the  disintegra- 
tion of  the  primary  cleavage  products.  No  matter  what  method 
is  used  to  decompose  a  given  protein  molecule,  the  primary  products 
are  largely  the  same  under  all  conditions.* 

In  the  process  of  hydrolysis  the  protein  molecule  is  gradually 
broken  down  and  less  complicated  aggregates  than  the  original  mole- 
cule are  formed,  which  are  known  as  proteoses,  peptones,  and  peptides, 
and  which  still  possess  true  protein  characteristics.  Further  hydrolysis 
causes  the  ultimate  transformation  of  these  sub.stances,  of  a  protein 
nature,  into  the  amino  acids  of  known  chemical  struclure.  In  this 
decomposition   the  protein  molecule  is  not  broken   flown  in  a  regular 

'  Alkaline  hydrolysis  yiulrjs  urea  .ind  ornilliinr  wliir.li  result  from  iiyi^ininr,  the  prodiK  t 
of  iic'ul  hvdrolvsis. 


PROTEINS.  63 

manner  into  1/2,  1/4,  1/8  portions  and  the  amino  acids  formed  in  a 
group  at  the  termination  of  the  hydrolysis.  On  the  contrary,  certain 
amino  acids  are  formed  very  early  in  the  process,  in  fact  while  the  main 
hyc^rolytic  action  has  proceeded  no  further  than  the  proteose  stage. 
Gradually  the  complexity  of  the  protein  portion  undergoing  decom- 
position is  simplified  by  the  splitting  off  of  the  amino  acids  and  finally 
it  is  so  far  decomposed  through  previous  cleavages  that  it  yields  only 
amino  acids  at  the  succeeding  cleavage.  In  short,  the  general  plan  of 
the  hydrolysis  of  the  protein  molecule  is  similar  to  the  hydrolysis  of 
starch.  In  the  case  of  starch  there  is  formed  a  series  of  dextrins  of 
gradually  decreasing  complexity  and  coincidently  with  the  formation 
of  each  dextrin  a  small  amount  of  sugar  is  split  off  and  finally  nothing 
but  sugar  remains.  In  the  case  of  protein  hydrolysis  there  is  a  series 
of  proteins  of  gradually  decreasing  complexity  produced  and  coinci- 
dently with  the  formation  of  each  new  protein  substance  amino  acids 
are  split  off  and  finally  the  sole  products  remaining  are  amino  acids. 
Inasmuch  as  diversity  in  the  method  of  decomposing  a  given 
protein  does  not  result  in  an  equally  diversified  line  of  decomposition 
products,  but,  on  the  other  hand,  yields  products  which  are  quite 
comparable  in  character,  it  may  be  argued  that  there  are  probably 
well-defined  lines  of  cleavage  in  the  individual  protein  molecule  and 
that  no  matter  what  the  force  brought  to  bear  to  tear  such  a  mole- 
cule apart,  the  disintegration,  when  it  comes,  will  yield  in  every  case 
certain  definite  fragments.  These  fragments  may  be  called  the  "  build- 
ing stones"  of  the  protein  molecule,  a  term  used  by  some  of  the  German 
investigators.  Take,  for  example,  the  decomposition  of  protein  which 
may  be  brought  about  through  the  action  of  the  enzyme  trypsin  of  the 
pancreatic  Juice.  When  this  enzyme  is  allowed  to  act  upon  a  given 
protein,  the  latter  is  disintegrated  in  a  series  of  definite  cleavages, 
resulting  in  the  formation  of  proteoses,  peptones,  and  peptides  in  regular 
order,  the  peptides  being  the  last  of  the  decomposition  products  which 
possess  protein  characteristics.  They  are  all  built  up  from  amino  acids 
and  are  therefore  closely  related  to  these  acids  on  the  one  side  and  to 
peptones  on  the  other.  We  have  di-,  tri-,  tetra-,  penta-,  deca-,  and 
poly-peptides  which  are  named  according  to  the  number  of  amino 
acids  included  in  the  peptide  molecule.  Following  the  peptides 
there  are  a  diverse  assortment  of  monamino  and  diamino  acids  which 
constitute  the  final  products  of  the  protein  decomposition.  These 
acids  are  devoid  of  any  protein  characteristics  and  are  therefore  decid- 
edly different  from  the  original  substance  from  which  they  were  derived. 
From  a  protein  of  huge  molecular  weight,  a  typical  colloid,  perhaps 


64  PHYSIOLOGICAL    CHEMISTRY. 

but  slightly  soluble,  and  entirely  non-diffusible,  we  have  passed  bv 
way  of  proteoses,  peptones,  and  peptides  to  a  class  of  simpler 
crystalline  substances  which  are,  for  the  most  part,  readily  soluble 
and  dillusible. 

These  amino  acids  after  their  production  in  the  process  of  diges- 
tion, as  just  indicated,  are  synthesized  within  the  organism  to  form 
protein  material  which  goes  to  build  up  the  tissues  of  the  body.  Jt 
is  thus  seen  that  the  amino  acids  are  of  prime  importance  in  the  animal 
economy.  Moreover,  it  is  important  to  remember  that  these  essential 
factors  in  metabolism  and  nutrition  cannot  be  produced  within  the 
animal  organism  from  their  elements,  but  are  only  yielded  upon  the 
hydrolysis  of  ingested  protein  of  animal  or  vegetable  origin. 

When  we  examine  the  formulas  of  the  principal  members  of  the 
crystalline  end-products  of  protein  decomposition  we  note  that  they 
are  invariably  acids,  as  has  already  been  mentioned,  and  contain  an 
NHj  group  in  the  ct  position.  This  relation  of  the  NHj  group  to 
the  acid  radical  is  constant,  no  matter  what  other  groups  or  radicals 
are  present.  We  may  have  straight  chains  as  in  alanine  and  glutamic 
acid,  the  benzene  ring  as  in  phenylalanine,  or  we  may  have  sulphurized 
bodies  as  in  cystine  and  still  the  formula  is  always  of  the  same  type,  i.  c, 

R— CH— COOH 

It  is  seen  that  this  characteristic  grouping  in  the  amino  acid  pro- 
vides each  one  of  these  ultimate  fragments  of  the  protein  molecule 
with  both  a  strong  acid  and  a  strong  basic  group.  For  this  reason 
it  is  theoretically  possible  for  a  large  number  of  these  amino  acids 
to  combine  and  the  resulting  combinations  may  be  very  great  in  num- 
ber, since  there  is  such  a  varied  assortment  of  the  acids.  The  pro- 
tein molecule,  which  is  of  such  mammoth  proportions,  is  probably 
constructed  on  a  foundation  of  this  sort.  Of  late  much  valuable 
data  have  been  collected  regarding  the  synlhclic  |)r()(luction  of  protein 
substances,  the  leaders  in  this  line  of  investigation  being  Fischer  and 
/\bderhalden.  After  having  gathered  a  mass  of  data  regarding  the 
final  products  of  l!ic  jjrotein  decomposition  and  demonstrating  thai 
amino  acids  were  the  ultimate  results  of  the  various  forms  of  decom- 
position, these  investigators,  and  notably  Fischer,  set  about  in  an 
effort  to  form,  from  these  amino  acids,  by  synthetic  means,  substances 
which  should  possess  protein  ( haracteristics.     Tlic  sim|)Iest  of  these 


PROTEINS.  65 

bodies  formed  in  this  way  was  synthesized  from  two  molecules  of 
glycocoll  with  the  liberation  of  water,  thus : 


CH,— NH3— CO    OH  H   HN— CH2— COOH. 

The  body  thus  formed  is  a  dipeptide,  called  glycyl-glycine.  In  an 
analogous  manner  may  be  produced  leucyl-leucine,  through  the  synthe- 
sis of  two  molecules  of  leucine  or  leucyl-alanyl- glycine  through  the 
union  of  one  molecule  of  leucine,  one  of  alanine,  and  one  of  glycocoll. 
By  this  procedure  Fischer  and  his  pupils  have  been  able  to  make  a 
large  number  of  peptides  containing  varied  numbers  of  amino-acid 
radicals,  the  name  polypeptides  being  given  to  the  whole  group  of 
synthetic  substances  thus  formed.  The  most  complex  polypeptide 
yet  produced  is  one  containing  fifteen  glycocoll  and  three  leucine 
residues. 

Notwithstanding  the  fact  that  most  synthetic  polypeptides  are 
produced  through  a  union  of  amino  acids  by  means  of  their  imide 
bonds,  it  must  not  be  imagined  that  the  protein  molecule  is  constructed 
from  amino  acids  linked  together  in  straight  chains  in  a  manner 
analogous  to  the  formation  of  simple  peptides,  such  as  glycyl-glycine. 
The  molecular  structure  of  the  proteins  is  much  too  complex  to  be 
explained  upon  any  such  simple  formation  as  that.  There  must  be 
a  variety  of  linkings,  since  there  is  a  varied  assortment  of  decomposi- 
tion products  of  totally  different  structure. 

Many  of  these  synthetic  bodies  respond  to  the  biuret  test,  are 
precipitated  by  phosphotungstic  acid,  and  behave,  in  other  ways, 
as  to  leave  no  doubt  as  to  their  protein  characteristics.  For  instance, 
a  number  of  amino  acids  each  possessing  a  sweet  taste  have  been 
synthesized  in  such  a  manner  as  to  yield  a  polypeptide  of  hitter  taste, 
a  well  known  characteristic  of  peptones.  From  the  fact  that  the 
polypeptides  formed  in  the  manner  indicated  have  free  acidic  and 
basic  radicals  we  gather  the  explanation  of  the  amphoteric  character 
of  true  proteins.  Fischer  expresses  the  encouraging  belief  that  he 
will  soon  be  able  to  produce  true  protein  by  the  synthesis  of  its  decom- 
position products.  Silk  fibroin  is  the  protein  substance  he  expects 
to  synthesize.  He  no  doubt  will  perform  this  joint  office  for  organic 
and  physiological  chemistry  if  it  is  capable  of  performance  by  the 
present  methods  of  technique.  Even  Fischer,  however,  is  frank  enough 
to  say  that  the  production  of  the  great  body  of  protein  substances 
synthetically,  will,  under  the  most  encouraging  conditions,  be  a  terrific 
task,  involving  the  ''life-work  of  a  whole  army  of  inventive  and  diligent 
chemists." 


66  PHYSIOLOGICAL    CHEMISTRY. 

For  the  benefit  of  those  especially  interested  in  such  matters  a 
photograph  of  the  Fischer  apparatus  (Fig.  22,  page  70)  used  in  the 
fractional  distillation,  /;/  vacuo,  of  the  esters  of  the  decomposition 
products  of  the  proteins,  as  well  as  micro-photographs  and  drawings 
of  preparations  of  several  of  these  decomposition  products  (Figs.  19 
to  31,  pages  67  to  79)  are  introduced.  For  the  preparations  and  the 
photograph  of  the  apparatus  the  author  is  indeljted  to  Dr.  T.  B.  Os- 
borne, of  Xew  Haven,  Conn.,  who  has  made  many  important  obser- 
\ations  upon  the  hydrolysis  of  proteins.  The  reproduction  of  the 
crystalline  form  of  some  of  the  more  recent  of  the  products  may  be 
of  interest  to  those  viewing  the  field  of  physiological  chemistry  from 
other  than  the  student's  aspect. 

An  extended  discussion  of  the  \arious  decomposition  products 
being  out  of  place  in  a  book  of  this  character,  we  will  simply  make 
a  few  general  statements  in  connection  with  the  primary  decom- 
position products. 

DISCUSSION  OF  THE  PRODUCTS. 

Ammonia,  XH3. — Ammonia  is  an  important  decomposition 
product  of  all  proteins  and  probably  arises  from  an  amide  group 
combined  with  a  carboxyl  group  of  some  of  the  amino  acids.  It 
is  possible  that  the  dibasic  acids,  aspartic  and  glutamic,  furnish  most 
of  these  carboxyl  groups.  This  is  indicated  by  the  more  or  less  close 
relationship  which  exists  between  the  amount  of  ammonia  and  that 
of  the  dibasic  acids  which  the  several  proteins  yield  upon  decomposi- 
tion. The  elimination  of  the  ammonia  from  proteins  under  the  action 
of  acids  and  alkalis  is  very  similar  to  that  from  amides  like  asparagine. 

Gycocoll,  C,H,,N02. — Glycocoll,  or  amino  acetic  acid,  is  the 
simplest  of  the  amino  acids  and  has  the  following  formula: 

NH, 

I 
H— C— COOH. 

H 

Glycocoll,  as  the  formula  shows,  contains  no  asymmetric  carbon  atom, 
and  is  the  only  amincj  acid  yielded  by  j)rolein  decomposition  which 
is  optically  inactive,  (jjycocoll  and  leucine  were  the  first  decom- 
position products  of  proteins  to  be  discovered  (1820).  Upon  ad- 
ministering benzoic  acid  to  animals  the  output  of  hii)puric  acid  in 
the  urine  is  greatly  increased,  thus  showing  a  synthesis  of  benzoic 


PROTEINS. 


67 


acid  and  glycocoll  in  the  organism  (see  p.  157,  Chapter  IX).  Glyco- 
coll,  ingested  in  small  amount,  is  excreted  in  the  urine  as  urea, 
whereas  if  administered  in  excess  it  appears  in  part  unchanged  in  the 
urine.  It  is  usually  separated  from  the  mixture  of  protein  decom- 
position products  as  the  hydrochloride  of  the  ester.  The  crystalline 
form  of  this  compound  is  shown  in  Fig.  19. 


Fig.  19. — Glycocoll  Ester  Hydrochloride. 

^  Alanine,  CgH^NO,. — Alanine  is  a-amino- propionic  acid,  and  as 
such  it  may  be  represented  structurally  as  follows: 

H     NH. 

H— C— C— COOH. 

H    H 

Obtained  from  protein  substances,  alanine  is  dextro-rotatory,  is  A'ery 
soluble  in  water,  and  possesses  a  sweet  taste.  Tyrosine,  phenylalanine, 
cystine,  and  serine  are  derivatives  of  alanine.  This  amino  acid  has  been 
obtained  from  nearly  all  proteins  examined.  Its  absence  from  those 
proteins  from  which  it  has  not  been  obtained  has  not  been  proven. 
Most  proteins  yield  relatively  small  amounts  of  alanine. 

Serine,    C3H7NO3. — Serine    is    a-amino- ^-hydroxy-propionic   acid 
and  possesses  the  following  structural  formula : 

OH  NH. 

1         I 
H-C  — C-COOH 

H      H 


68 


PHYSIOLOGICAL    CHEMISTRY. 


Serine  obtained  from  proteins  is  laevo-rotalory,  possesses  a  sweet 
taste,  and  is  quite  soluble  in  water.  Serine  is  not  obtained  in  quantity 
from  most  proteins,  but  is  yielded  abundantly  by  silk  glue.  Owing 
10  the  difficulty  of  separating  serine  it  has  not  been  found  in  a  number 
of  proteins  in  which  it  probably  occurs.  Serine  crystals  are  shown  in 
Fiy.  20.  below. 


Fig.  20. — Serine. 


Phenylalanine,    C9HjjN02. — This    product    is   phenyl-a-amvio- 
propionk  acid,  and  may  be  represented  graphically  as  follows: 


H    NH^ 

I       I 
/\ C-C-COOH. 


The  la:\o-rotatory  form  is  (obtained  from  jjroteins.  Phenylalanine 
has  been  obtained  from  all  the  proteins  examined  except  from  the 
protamines  and  some  of  the  albuminoids.  'J'hc  yield  of  this  body 
from  the  decomposition  of  jjroteins  is  fre(jueotly  greater  than  the 
yield  of  tyrosine.  The  crystalline  form  of  j)henylalanine  is  shown 
in  P'ig.  21,  p.  ()(). 

Tyrosine,  ('„Ii,,N03. — Tyrosine,  one  of  the  first  discovered  end- 
products  of  j^rotein  decomposition,  is  the  amino  acid,  p-oxyphrnyl- 
«!- amino  propionic  acid.    It  has  the  following  formula : 


PROTEINS. 
H      NH,, 

~C-C-COOH. 


69 


OH 


The  tyrosine  which  resuhs  from  protein  decomposition  is  usually 
Isevo-rotatory  although  the  dextro-rotatory  form  sometimes  occurs. 
Tyrosine  is  one  of  the  end-products  of  tryptic  digestion  and  usually 


Fig.  21. — PHENrYLAL.\NiNi;. 


separates  in  conspicuous  amount  early  in  the  process  of  digestion. 
It  does  not  occur,  however,  as  an  end-product  of  the  decomposition 
of  gelatin. 

Tyrosine  is  found  in  old  cheese,  and  derives  its  name  from  this 
fact.  It  crystallizes  in  tufts,  sheaves,  or  balls  of  fine  needles,  which 
decompose  at  295°  C.  and  are  sparingly  soluble  in  cold  (1-2454) 
water,  but  much  more  so  in  boiling  (1-154)  water.  Tyrosine  forms 
soluble  salts  with  alkalis,  ammonia,  or  mineral  acids,  and  is  soluble, 
with  difficulty,  in  acetic  acid.  It  responds  to  Millon's  reaction,  thus 
showing  the  presence  of  the  hydroxyphenyl  group,  but  gives  no  other 
protein  test.  The  aromatic  groups  present  in  tyrosine,  phenyl-alanine, 
and  tryptophane  cause  proteins  to  yield  a  positive  xanthoproteic 
reaction.  In  severe  cases  of  typhoid  fever  and  smallpox,  in  acute 
}'ellow  atrophy  of  the  liver,  and  in  acute  phosphorus  poisoning,  tyro- 
sine has  been  found  in  the  urine.  Tyrosine  crystals  are  shown  in 
Fig.  23,  p.  71. 


PHYSIOLOGICAL    CHEMISTRY. 


Fig.  22. — l-ibCUER  Ain'.\R.\TVs. 
Reproduced  from  a  photograph  made  by  Prof.   E.  T.   Reichert,  of  the  Uiiversiiy  of 
Pennsylvania.     The  negative  was  furnished   h\'   Dr.   T.    B.   Osborne,   of  Xew  H;nen, 
Conn. 

A,  Tank  into  which  freezing  mixture  is  jjumpcd  and  from  wliirli  it  tlows  tlirough  the 
condenser,  B;C,  flask  from  whi(  ii  the  esters  are  distilled,  the  di'^tillalc  being  collected  in  D; 
E,  a  Dewar  tlask  containing  liquitl  air  serving  as  a  cooler  for  condensing  tube  /•",■  (.!  and  C, 
tubes  leading  to  the  (lerycU  jjump  by  which  the  vacuum  is  maintained;  /,  tulie  leading  to  a 
McLef)fl  gauge  (not  shown  in  figure);  ./,  a  bath  containing  freezing  mi.xture  in  which  the 
receiver  D  is  immersed;  K,  a  bath  of  water  during  the  first  part  of  the  distillation  and  of 
oil  during  the  last  ]iart  of  the  process;  1-5,  stop  cocks  which  permit  the  cutting  out  of 
flifTerent  parts  of  the  apparatus  as  the  |)roceflure  demands. 

Cystine,  C'dHjjO^NjS.,.-  Friedmann  has  recently  shown  cystine 
to  be  the  disulphide  of  a-amino-fi-l/iio/drlir  ncid^  and  to  possess  the 
followinj^  structural    formula: 

CH./S-S-CH., 

CHXH,.     CUNH., 

I  "      ' 

coon     cooii 


'.Also  (alli-d  'i-diamino- V-diihio-fliiai  t\lii    a(  id. 


PROTEINS.  71 

Cystine  is  the  principal  sulphur-containing  body  obtained  from 
the  decomposition  of  protein  substances.  It  is  obtained  in  greatest 
amount  as  a  decomposition  product  of  such  keratin-containing  tissues 


Fig.  22. — Tyrosine. 


as  horn,  hoof,  and  hair.  Cystine  occurs  in  small  amount  in  normal 
urine  and  is  greatly  increased  in  Cjuantity  under  certain  pathological 
conditions.     It  crystallizes  in  thin,  colorless,  hexagonal  plates  which 


Fig.  24. — Cysthste. 

are  shown  in  Fig.  24.  Cystine  is  very  slightly  soluble  in  water  but 
its  salts,  with  both  bases  and  acids,  are  readily  soluble  in  water. 
It  is  laevo-rotatory. 


/^ 


PHYSIOLOGICAL    CHEMISTRY. 


It  has  recently  been  claimed  that  cystine  occurs  in  two  forms, 
/.  e.,  stone-cystine  and  protein-cystine  and  thai  these  two  forms  are 
distinct  in  their  properties.    This  view  is  incorrect. 

For  a  discission  of  cystine  sediments  in  urine  see  Chapter  XX. 

Tryptophane,  Cj,Hj,N,02. — According  to  Ellinger,  tryptophane 
is  indol-oL-amino-propionic  acid.  Recently  Ellinger  and  FUimand  have 
shown  that  it  possesses  the  following  formula : 

/\ C  CH,  CHCNH,,)  COOH 


CH 
NH 

Tryptophane  is  the  mother -substance  of  indole,  skatole,  skatole 
acetic  acid  and  skatole  carboxylic  acid,  all  of  which  are  formed  as 
secondary  decomposition  products  of  proteins.  Its  presence  in  pro- 
tein substances  may  be  shown  by  means  of  the  Adamkiewicz  reaction 
(jr  the  Hopkins-Cole  reaction  (see  page  89).  It  may  be  detected  in  a 
tryptic  digestion  mixture  through  its  property  of  giving  a  violet  color- 
reaction  with  bromine  water.  Tryptophane  is  yielded  by  nearly  all 
proteins,  but  has  been  shown  to  be  entirely  absent  from  zein,  the  pro- 
lamin  (alcohol-soluble  protein)  of  maize. 

Solutions  of  tryptophane  in  sodium  hydroxide  are  dextro-rotatory. 
Upon  being  heated  to  266°  C.  tryptophane  decomposes  with  the  cvo- 
hition  of  gas. 

Histidine,  C^HgNgOg. — Histidine  is  a-amino-^-imidazol-propionic 
arid  with  the  following  structural  formula: 

H    NH2 

I       I 
HC  =   C-C-C-COOH. 

I      '       I 
!      H    H 

HNX/N 

CH 

The  histidine  obtained  from  ])roteins  is  laivo  rotatory.  It  has 
been  obtained  from  all  the  proteins  thus  far  examined,  the  majority 
of  Ihem  yielding  about  2.5  j)er  cent  of  the  amino  acid.  However, 
about  I  r  per  cent  was  obtained  by  Abderjialden  from  globin,  the  pro- 
tein constituent  of  oxyha-moglobin  and  about  13  per  cent  by  Kossel 
and  Kutscher  from  the  protamine  sturine. 


PROTEINS.  73 

Crystals  of  histidine  dichloride  are  shown  in  Fig.  25,  below. 

Knoop^s  Color  Reaction  for  Histidine. — To  an  aqueous  solution 
of  histidine  or  a  histidine  salt  in  a  test-tube  add  a  little  bromine  water. 
A  yellow  coloration  develops  in  the  cold  and  upon  further  addition 
of  bromine  water  becomes  permanent.  If  the  tube  be  heated/  the  color 
will  disappear  and  will  shortly  be  replaced  by  a  faint  red  coloration 
which  gradually  passes  into  a  deep  wine  red.  Usually  black,  amor- 
phous particles  separate  out  and  the  solution  becomes  turbid. 


Fig.  25. — Histidine  Bichloride. 

The  reaction  cannot  be  obtained  in  solutions  containing  free  alkali. 
It  is  best  to  use  such  an  amount  of  bromine  as  will  produce  a  per- 
manent yellow  color  in  the  cold.  The  use  of  a  less  amount  of  bromine 
than  this  produces  a  weak  coloration  whereas  an  excess  of  bromine 
prevents  the  reaction.  The  test  is  not  very  delicate,  but  a  character- 
istic reaction  may  always  be  obtained  in  i  :  1000  solutions.  The  only 
histidine  derivative  which  yields  a  similar  coloration  is  imidazolethyl- 
amine,  and  the  reaction  in  this  case  is  rather  weak  as  compared  with 
the  color  obtained  with  histidine  or  histidine  salts. 

Valine,  CgHj^NOg- — The  amino-va*lerianic  acid  obtained  from 
proteins  is  <x-amino-isovalerianic  acid,  and  as  such  bears  the  following 
formula: 

CH3    NH2 

!        I 
H-C C-COOH. 


CH3    H 

'  The  same  reaction  will  take  olace  in  the  cold  more  slowly. 


74  PHYSIOLOGICAL    CHEMISTRY. 

It  closely  resembles  leucine  in  many  of  its  properties,  but  is  more 
soluble  in  water.  It  is  a  diflicult  matter  to  identify  \aline  in  the 
presence  of  leucine  and  isoleucine  inasmuch  as  these  amino  acids 
crystallize  together  in  such  a  way  that  the  combination  persists  even 
after  repeated  recrystallizations.    \'aline  is  dextro-rotatory. 

Arginine,  CgHj^N^O,. — Arginine  is  gnanidiue.-a-amino-valerianic 
acid  and  possesses  the  following  structural  formula : 

H    H    H    NH., 

i         1         ';         I 

NH-C-C-C-C-COOH. 

NH=C        H    H    H    H 

NH2 

It  has  been  obtained  from  every  protein  so  far  subjected  to  decom- 
position. The  arginine  obtained,  from  proteins  is  dextro-rotatory, 
and  has  pronounced  basic  properties,  reacts  strongly  alkaline  to 
litmus,  and  forms  stable  carbonates.  Because  of  these  facts,  some 
investigators  consider  arginine  to  be  the  nucleus  of  the  protein  mole- 
cule. It  is  obtained  in  widely  different  amounts  from  different  proteins, 
over  85  per  cent  of  certain  protamines  having  been  obtained  in  the 
form  of  this  amino  acid.  It  is  claimed  that  in  the  ordinary  metabolic 
activities  of  the  animal  body  arginine  gi\es  rise  to  urea.  While  this 
claim  is  probably  true,  it  should,  at  the  same  time,  be  borne  in  mind 
that  the  greater  part  of  the  protein  nitrogen  is  eliminated  as  urea  and 
that,  therefore,  but  a  very  small  part  can  arise  from  arginine. 

Leucine,  CgH,3N02. — Leucine  is  an  abundant  end-product  of 
the  decomposition  of  protein  material,  and,  together  with  glycocoll, 
was  the  first  of  these  products  to  be  discovered  (1820).  It  is  ix-amhw- 
isobulyl-acelir  urid,  and  therefore  has  the  following  formula: 

cn.       Nil, 

CHCH.,C-COOH. 

I  '    I 

CH,,  H 

'J'he  leucine  which  results  from  ])rolein  (lcc()m])osilioii  is  /-leucine. 
Leucine  is  present  normally  in  the  jjancreas,  ihyiuiis,  Ihyroid,  sj)leen, 
brain,  liver,  kidneys,  and  salivary  glands.  It  has  been  found  palh- 
nloj^ically  in  the  urine  fin  acute  yellow  atrophy  of  the  liver,  in  acute 
|;hosj)horus  poisoning,  and  in  severe  cases  of  ty])hoid  fever  and  small- 
jjoxj,  anrl  in  the  li\er,  blo(Kl,  and  jjus. 


PROTEINS. 


75 


Pure  leucine  crystallizes  in  thin,  white,  hexagonal  plates.  Crystals 
of  pure  leucine  are  reproduced  in  Fig.  26.  It  is  rather  easily  soluble 
in  water  (46  parts),  alkalis,  ammonia,  and  acids.  On  rapid  heating 
to  295°  C,  leucine  decomposes  with  the  formation  of  carbon  dioxide, 
ammonia,  and  amylamine.  Aqueous  solutions  of  leucine  obtained 
from  proteins  are  laevo-rotatory,  but  its  acid  or  alkaline  solutions  are 
dextro-rotatory.     So-called    impure   leucine^    is   a   slightly   refracti^"e 


Fig.  26. — Leucine. 

substance,  which  generally  crystallizes  in  balls  having  a  radial 
structure,  or  in  aggregations  of  spherical  bodies,  Fig.  104,  Chapter 
XX. 

Isoleucine,    CgHj3N02. — Isoleucine  is    a-amino-methyl-ethyl-pro- 
pionic  acid  J  and  possesses  the  following  structural  formula: 

CH3     NH3 


CH 


C.H. 


C— COOH. 
H 


This  amino  acid  was  recently  discovered  by  Ehrlich.  Its  presence 
has  been  established  among  the  decomposition  products  of  only  a 
few  proteins  although  it  probably  occurs  among  those  of  many  or 
most  of  them.  Ehrlich  has  shown  that  the  (/-amy!  alcohol  which 
is  produced  by  yeast  fermentation  originates  from  isoleucine  and 
the  isoamylalcohol  originates  from  leucine.  Isoleucine  is  dextro- 
rotatory. 

'  These  balls  of  so-called  impure  leucine  do  contain  considerable  leucine,  but  inasmuch 
as  they  may  contain  many  other  things  it  is  a  bad  practice  to  allude  to  them  as  leucine. 


yO  PHYSIOLOGICAL    CHEMISTRY. 

Lysine,  CgHj^XjO,. — The  three  bodies,  lysine,  arginine,  and  his- 
tidine,  are  frequently  classed  together  as  the  hexone  bases.  Lysine 
was  the  first  of  the  bases  discovered.  It  is  a-e-diamino-caproic  acid 
and  hence  possesses  the  following  structure: 

NH,  H     H     H     NH., 

I    '  !      i      1     I 

H-C  -  C-C-C-C-COOH 
H      H     H      H     H 

It  is  dextro-rotatory  and  is  found  in  relatively  large  amount  in  casein 
and  gelatin.  Lysine  is  obtained  from  nearly  all  proteins,  but  is  absent 
from  the  vegetable  proteins  which  arc  soluble  in  strong  alcohol.     It 


I'ic.  27. — Lysine  Picrate. 

is  the  mother-substance  of  cadaverin  and  has  ne\er  been  obtained 
in  crystalline  form.  Lysine  is  usually  obtained  as  the  picrate  which 
is  sparingly  soluble  in  water  and  crystallizes  readily.  These  crystals 
are  shown  in  Fig.  27. 

Aspartic  Acid,  CJ^^^NO^. — Aspartic  acid   is  amino-succinic  acid 
and  has  the  following  structural  formula: 

NH3 

I 
CHCOOH 

I 
CH./C()()I1. 


PROTEINS. 


// 


The  amide  of  aspartic  acid,  asparagine,  is  very  widely  distributed  in 
the  vegetable  kingdom.  The  crystalline  form  of  aspartic  acid  is 
exhibited  in  Fig.   28. 

Aspartic  acid  has  been  found  among  the  decomposition  products 
of  all  the  proteins  examined,  except  the  protamines.  It  has  not  been 
obtained,  however,  in  v^ery  large  proportion  from  any  of  them.  The 
aspartic  acid  obtained  from  protein  is  laevo-rotatory. 


Fig.  28. — Aspartic  Acid. 

Glutamic  Acid,  CgHgNO^. — This  acid  is  a-amino-normal-glutaru 
acid  and  as  such  bears  the  following  graphic  formula: 

NH, 

CH-COOH 

CH, 


CH,-COOH. 

Glutamic  acid  is  yielded  by  all  the  proteins  thus  far  examined, 
except  the  protamines,  and  by  most  of  these  in  larger  amount  than 
any  other  of  their  decomposition  products.  It  is  yielded  in  especially 
large  proportion  by  most  of  the  proteins  of  seeds,  41.32  per  cent 
having  been  obtained  very  recently  by  Kleinschmitt  from  the  hydrolysis 
of  hordein,  the  prolamin  of  barley.  This  is  the  largest  amount  of  any 
single  decomposition  product  yet  obtained  from  any  protein  except 
the  protamines.^ 

'  Up  to  this  time  the  j-ield  of  37.33  per  cent  obtained  by  Osborne  and  Harri.^  from 
sfliadin  of  wheat  was  the  maximum  vield. 


78 


PHYSIOLOGICAL    CHEMISTRY. 


Glutamic  acid  and  aspartic  acid  are  the  only  dibasic  acids  which 
have  thus  far  been  obtained  as  decomposition  products  of  proteins. 
As  there  is  an  apparent  relation  between  the  proportion  of  these 
acids  and  that  of  ammonia  which  the  different  proteins  yield  it  is 
possible  that  one  of  the  carboxyl  groups  of  these  acids  is  united  with 
XH,  as  an  amide,  the  other  carboxyl  group  being  united  in  poly- 


Fig.  2q. — Glutamic  Acid. 
Reproduced  from  a  micro-photograph  made  by  Prof.  E.  T.  Reichert,  of  the  University  of 

Pennsylvania. 

peptide  union  (see  page  64)  with  some  other  amino  acid.    This  might 
Ije  represented  by  the  following  formula: 

R-CHNH-COOH 


CO  -  CHNH2 -  CH2-  CH2-  CONH,. 

It  has  not  been  definitely  proven,  however,  that  this  form  of  linking 
actually  occurs. 

The  glutamic  acid,  yielded  by  proteins  upon  hydrolysis,  is  dextro- 
rotatory.   Crystals  of  glutamic  acid  are  reproduced  in  Fig.  29,  above. 

Proline,  C^H^NOj. — Proline  is  ct-pyrrolidine-carboxylic  acid  and 
possesses  the  following  graphic  structure: 


H,C 


CH, 


H^C  x/CHCOOII. 
NH 

Proline  was  first  (obtained  as  a  decomposition  product  of  casein.  Pro- 
line obtained  from  ])roteins  is  laevo-rotatory  and  is  the  only  protein 
decompositifjn  jjroduct  which  is  readily  soluble  in  alcohol,     it  is  also 


PROTEINS. 


79 


one  of  the  few  heterocyclic  compounds  obtained  from  proteins.  Pro- 
line was  quite  recently  discovered,  but  has  since  been  found  among 
the  decomposition  products   of  all  proteins  except   the  protamines. 


Fig. 


-L.EVO-a-PROLINE. 


The  maximum  yield  reported  is  13.73  P^^  cent  obtained  by  Osborne 
and  Clapp  from  the  hydrolysis  of  hordein.  More  recently  Fischer  and 
Boehner^  reported  having  obtained  7.7  per  cent  from  the  hydrolysis 


Fig.  31. — Copper  Salt  of  Proline. 
Reproduced  from  a  micro-photograph  made  by  Prof.  E.  T.  Reichert,  of  the  University  of 

Pennsylvania. 

of  gelatine.  The  crystalline  form  of  I avo- a- proline  is  shown  in  Fig.  30, 
and  the  copper  salt  of  proline  is  represented  by  a  micro-photograph  in 
Fig.  31,  above.    The  crystals  of  the  copper  salt  have  a  deep  blue  color 

'  Fischer  and  Boehner:     Zeit.  phys.  chem.,  65,  p.  118,  1910. 


So  PHYSIOLOGICAL    CHEMISTRY. 

but  when  they  lose  their  water  of  crystallization  they  assume  a  char- 
acteristic violet  color. 

Oxyproline,  C5HyX03. — Oxyproline  was  recently  discovered  by 
Fischer.  It  has  as  yet  been  obtained  from  only  a  few  proteins,  but 
this  may  be  due  to  the  fact  that  only  a  few  have  been  examined  for 
its  presence.    Its  structure  has  not  yet  been  established. 

Diaminotrihydroxydodecanoic  Acid,  CjjHjgNjOg. — This  amino 
acid  was  disco\ered  by  Fischer  and  Abderhalden  as  a  product  of 
the  hydrolysis  of  casein.  It  has  thus  far  been  obtained  from  no  other 
source.    It  is  ke\o-rotatorv  and  its  constitution  has  not  been  determined. 


Experiments. 

While  the  ordinary  courses  in  physiological  chemistry  preclude 
any  extended  study  of  the  decomposition  products  of  proteins,  the 
manipulation  of  a  simple  decomposition  and  the  subsequent  isola- 
tion and  study  of  a  few  of  the  products  most  easily  and  quickly  ob- 
tained will  not  be  without  interest.^  To  this  end  the  student  may 
use  the  following  decomposition  procedure:  Treat  the  protein  in  a 
large  flask  with  water  containing  3-5  per  cent  of  HjSO^  and  place 
it  on  a  water-bath  until  the  protein  material  has  been  decompose'd 
and  there  remains  a  fine,  fluffy,  insoluble  residue.  Filter  off  this 
residue  and  neutralize  the  filtrate  with  Ba(0H)2and  BaCOj.  Filter 
off  the  precipitate  of  BaSO^  which  forms  and  when  certain  that 
the  fluid  is  neutral  or  faintly  acid,"  concentrate  (first  on  a  wire  gauze 
and  later  on  a  water-bath)  to  a  syrup.  This  syrup  contains  the  end 
products  of  the  decomposition  of  the  protein,  among  which  are  pro- 
teoses, peptones,  tyrosine,  leucine,  etc.  Add  95  per  cent  alcohol  slowly 
to  the  warm  syrup  until  no  more  precipitate  forms,  stirring  continu- 
ously with  a  glass  nxJ.  This  precipitate  consists  of  proteoses  and 
peptones.  Gather  the  sticky  ])recipitate  on  the  rod  or  the  sides  of  the 
flish,  and,  after  warming  the  solution  gently  for  a  few  moments,  filter 
it  through  a  filter  pajjcr  which  has  not  been  previously  moistened. 
.After  dissolving  the  j)recipitate  of  ])r()teoses  and  ])ej)tones  in  water'' 

'  The  procedure  here  set  forth  li;is  nothing;  in  comiiioii  witli  llu-  proiedurc  by  means 
of  which  the  long  line  (jf  deconijiosition  jirodiicls  just  enumerated  are  obtained.  This 
latter  process  is  an  exceedingly  complicated  one  \vlii(  h  is  entirely  outside  the  province  of 
any  course  in  physiologic  al  chemistry. 

*  If  the  scjjution  is  alkaline  in  reaction  at  this  puinl,  llu-  ainiim  iirids  will  be  Ijroken 
down  anrl  ammcmia  will  be  cv(jlved. 

*  At  this  |X)int  the  af|UCOus  s<jlution  of  the  proteoses  and  |jc|)i<)nc.';  may  J)C  filteref!  to 
remove  any  JlaSO,  which  may  still  remain.  Tyrosine  crystals  will  also  be  found  here, 
since  it  is  less  soluble  than  the  lem  ine  ami  may  arlherc  to  the  proteose-peptone  precipitate. 
Afld  the  crystals  of  tynjsine  to  the  warm  ali  ohol  liltrate. 


PROTEINS.  61 

the  solution  may  be  treated  according  to  the  method  of  separation 
giv^en  on  page  112. 

The  leucine  and  tyrosine,  etc.,  are  in  solution  in  the  warm  alcoholic 
filtrate.  Concentrate  this  filtrate  on  the  water-bath  to  a  thin  syrup, 
transfer  it  to  a  beaker,  and  allow  it  to  stand  over  night  in  a  cool  place 
for  crystallization.  The  tyrosine  first  crystallizes  (Fig.  23,  page  71), 
followed  later  by  the  formation  of  characteristic  crystals  of  impure 
leucine  (see  Fig.  105,  Chapter  XX).  After  examining  these  crystals 
under  the  microcsope,  strain  off  the  crystalline  material  through  fine 
muslin,  heat  it  gently  in  a  little  water  to  dissolve  the  leucine  (the  tyro- 
sine will  be  practically  insoluble)  and  filter.  Concentrate  the  filtrate 
and  allow  it  to  stand  in  a  cool  place  over  night  for  the  crude  leucine 
to  crystallize.  Filter  off  the  crystals  and  use  them  in  the  tests  for 
leucine  given  on  page  82.  The  crystals  of  tyrosine  remaining  on  the 
paper  from  the  first  filtration  may  be  used  in  the  tests  for  tyrosine  as 
given  below.  If  desired,  the  tyrosine  and  leucine  may  be  purified  by 
recrystallizing  in  the  usual  manner.  Habermann  has  suggested  a 
method  of  separating  leucine  and  tyrosine  by  means  of  glacial  acetic 
acid. 

Experiments  on  Tyrosine. 

Make  the  following  tests  with  the  tyrosihe  crystals  already  pre- 
pared or  upon  some  pure  tyrosine  furnished  by  the  instructor. 

1.  Microscopical  Examination. — Place  a  minute  crystal  of  tyro- 
sine on  a  slide,  add  a  drop  of  water,  cover  with  a  coverglass,  and 
examine  microscopically.  Now  run  more  water  under  the  cover- 
glass  and  warm  in  a  bunsen  flame  until  the  tyrosine  has  dissolved. 
Allow  the  solution  to  cool  slowly,  then  examine  again  microscopically, 
and  compare  the  crystals  with  those  shown  in  Fig.  23,  page  71. 

2.  Solubility. — Try  the  solubility  of  very  small  amounts  of  tyro- 
sine in  cold  and  hot  water,  cold  and  hot  95  per  cent  alcohol,  dilute 
NH.OH,  dilute  KOH  and  dilute  HCl. 

3.  Sublimation. — Place  a  little  tyrosine  in  a  dry  test-tube,  heat 
gently  and  notice  that  the  material  does  not  sublime.  How  does  this 
compare  with  the  result  of  Experiment  3  under  Leucine  ? 

4.  Hoffman's  Reaction. — This  is  the  name  given  to  Millon's, 
reaction  when  employed  to  detect  tyrosine.  Add  about  3  c.c.  of  water 
and  a  few  drops  of  Millon's  reagent  to  a  little  tyrosine  in  a  test-tube. 
Upon  dissolving  the  tyrosine  by  heat  the  solution  gradually  darkens 
and  may  assume  a  dark  red  color.  What  group  does  this  test  show 
to  be  present  in  tyrosine  ? 

6 


82  PHYSIOLOGICAL    CHEMISTRY. 

5.  Piria's  Test. — Warm  a  little  tyrosine  on  a  watch  glass  on  a 
boiling  water-bath  for  20  minutes  with  3-5  drops  of  cone.  HjSO^. 
Tyrosine  sulphuric  acid  is  formed  in  the  process.  Cool  the  solution 
and  wash  it  into  a  small  beaker  with  water.  Now  add  CaCOg  in 
substance  slowly  with  stirring,  until  the  reaction  of  the  solution  is 
no  longer  acid.  Filter,  concentrate  the  filtrate,  and  add  to  it  a  few  drops 
(avoid  an  excess)  of  very  dilute  neutral  ferric  chloride.  A  purple  or 
violet  color,  due  to  the  formation  of  the  ferric  salt  of  tyrosine-sulphuric 
acid,  is  produced.  This  is  one  of  the  most  satisfactory  tests  for  the 
identification  of  tyrosine. 

6.  Morner's  Test, — Add  about  3  c.c.  of  Morner's  reagent^  to  a 
little  tyrosine  in  a  test-tube,  and  gently  raise  the  temperature  to  the 
boiling-point.     A  green  color  results. 

Experiments  on  Leucine. 

Make  the  following  tests  upon  the  leucine  crystals  already  pre- 
I>ared  or  upon  some  pure  leucine  furnished  by  the  instructor. 

I,  2  and  3.  Repeat  these  experiments  according  to  the  directions 
given  under  Tyrosine  (page  81). 

*  Morner's  .reagent  is  prepared  by  thoroughly  mi.xing  t  volume  of  fcirnialiii,  45  Nolunics 
of  distilled  water,  and  55  volumes  of  concentrated  sulphuric  acid. 


CHAPTER  V. 

PROTEINS:  THEIR  CLASSIFICATION  AND 
PROPERTIES. 

From  what  has  already  been  said  in  Chapter  IV  regarding  the 
protein  substances  it  will  be  recognized  that  the  grouping  of  the  diverse 
forms  of  this  class  of  substances  in  a  logical  manner  is  not  an  easy 
task.  The  fats  and  carbohydrates  may  be  classified  upon  the  funda- 
mental principles  of  their  stereo-chemical  relationships,  whereas  such 
a  system  of  classification  in  the  case  of  the  proteins  is  absolutely  im- 
possible since,  as  we  have  already  stated,  the  molecular  structure  of 
these  complex  substances  is  unknown.  Because  of  the  diversity  of 
standpoint  from  which  the  proteins  may  be  viewed,  relative  to  their 
grouping  in  the  form  of  a  logically  classified  series,  it  is  obvious  that 
there  is  an  opportunity  for  the  presentation  of  classifications  of  a 
widely  divergent  character.  The  fact  that  there  were  until  recently 
at  least  a  dozen  different  classifications  which  were  recognized  by 
various  groups  of  English-speaking  investigators  emphasizes  the  diffi- 
culties in  the  way  of  the  individual  or  individuals  who  would  offer 
a  classification  which  should  merit  universal  adoption.  Realizing 
the  great  handicap  and  disadvantage  which  the  great  diversity  of 
the  protein  classifications  was  forcing  upon  the  workers  in  this 
field,  the  Chemical  and  Physiological  Societies  of  England  recently 
drafted  a  classification  which  appealed  to  these  groups  of  scientists 
as  fulfilling  all  requirements  and  presented  it  for  the  consideration 
of  the  American  Physiological  Society  and  the  American  Society  of 
Biological  Chemists.  The  outcome  of  this  has  been  that  there  are  now 
only  two  protein  classifications  which  are  recognized  by  English-speak- 
ing scientists,  one  the  British  Classification,  the  other  the  American 
Classification.  These  classifications  are  very  similar  and  doubtless 
will  ultimately  be  merged  into  a  single  classification.  In  our  con- 
sideration of  the  proteins  we  shall  conform  in  all  details  to  the  American 
Classification.  In  this  connection  we  will  say,  however,  that  we  feel 
that  the  English  Societies  have  strong  grounds  for  preferring  the  use 
of  the  term  scleroproteins  for  albuminoids  and  chromo proteins  for 
haemoglobins.     The  two  classifications  are'  as  follows : 

83 


84  PHYSIOLOGICAL    CHEMISTRY. 

CLASSIFICATION  OF  PROTEINS  ADOPTED  BY  THE  AMERI- 
CAN PHYSIOLOGICAL  SOCIETY  AND  THE  AMERI- 
CAN SOCIETY  OF  BIOLOGICAL  CHEMISTS. 

I.  SIMPLE  PROTEINS. 

Protein  substances  which  yield  only  a-amino  acids  or  their  deri\a- 
tives  on  hydrolysis. 

(a)  Albumins. — Soluble  in  pure  water  and  coagulable  by  heat, 
e.  g.,  ovalbumin,  serum  albumin,  lactalbiimin,  vegetable  albumins. 

{b)  Globulins. — Insoluble  in  pure  water  but  soluble  in  neutral 
solutions  of  salts  of  strong  bases  with  strong  acids/  e.  g.,  serum  globulin, 
ovoglobulin,  edesdn,  amandin,  and  other  vegetable  globulins. 

(c)  Glutelins. — Simple  proteins  insoluble  in  all  neutral  solvents, 
but  readily  soluble  in  very  dilute  acids  and  alkalis,^  e.  g.,  glutenin. 

(d)  Alcohol-soluble  Proteins  (Prolamins)  .^ — Simple  proteins 
soluble  in  70-80  per  cent  alcohol,  insoluble  in  water,  absolute  alcohol, 
and  other  neutral  solvents/  e.  g.,  zein,  gliadin,  hordein,  and  bynin. 

(e)  Albuminoids. — Simple  proteins  possessing  a  similar  struc- 
ture to  those  already  mentioned,  but  characterized  by  a  pronounced 
insolubility  in  all  neutral  solvents/  e.  g.,  elastin,  collagen,  keratin. 

(/)  Histones. — Soluble  in  water  and  insoluble  in  very  dilute 
ammonia,  and,  in  the  absence  of  ammonium  salts,  insoluble  even  in 
excess  of  ammonia;  yield  precipitates  with  solutions  of  other  proteins 
and  a  coagulum  on  heating  which  is  easily  soluble  in  very  dilute  acids. 
On  hydrolysis  they  yield  a  large  number  of  amino  acids  among  which 
the  basic  ones  predominate.  In  short,  histones  are  basic  proteins 
which  stand  between  protamines  and  true  prx)teins,  e.  g.,  globin, 
thymus  histone,  scombrone. 

(g)  Protamines. — Simj)lcr  polypeptides  than  the  proteins  in- 
cluded in  the  preceding  grouj)s.  They  are  soluble  in  water,  uncoag- 
iilable  by  heat,  have  the  projjcrty  of  precipitating  aqueous  solutions 
of  other  proteins,   possess  strong  basic  properties  and   form  stable 

'The  precipitation  limits  with  ammonium  .sulphate  should  not  be  made  a  basis  for 
fhstinguishing  the  albumins  from  the  globulins. 

^  Such  substances  occur  in  abuiiflance  in  the  seeds  of  cereals  and  doubtless  represent  a 
\vell-<lefined  natural  group  of  sim];le  ])roteins. 

"The  name  prolamine  has  been  suggested  for  these  alcohol-soluble  proteins  by  Dr. 
7'homas  H.  Osborne  (Science,  1908,  XXVI 1 1,  p.  417).  It  is  a  very  titling  term  inasmuch 
as  ui>on  hyflrolysis  they  yield  particularly  large  amounts  of  proline  and  ammonia. 

*  The  subcl'ass<'S  defmefl  (a,  h,  c,  d,)  are  exemi)li(ied  by  proteins  obtained  from  both 
plants  and  animals.  The  use  of  approjiriate  |)relixes  will  sulTnc  to  indicate  the  origin  of 
the  compounds,  e.  f^.,  ovoglobulin,  la(  talbumin,  etc. 

■'  These  form  the  [jrincipal  organic  ( onstilncnts  of  the  skeletal  sliucturc  of  animals 
and  also  their  external  covering  and  lis  appendages.  This  defmition  docs  not  provide 
for  gelatin  which  is,  however,  an  arlilic  iai  cjcrivative  of  collageri. 


PROTEINS.  85 

salts  with  strong  mineral  acids.  They  yield  comparati\'ely  few  amino 
acids,  among  which  the  basic  ones  predominate.  They  are  the  simplest 
natural  proteins,  e.  g.,  salmine,  sturine,  clupeine,  scomhrine. 

II.  CONJUGATED  PROTEINS. 

Substances  which  contain  the  protein  molecule  united  to  some 
other  molecule  or  molecules  otherwise  than  as  a  salt. 

{a)  Nucleoproteins. — Compounds  of  one  or  more  protein  mole- 
cules with  nucleic  acid,  e.  g.,  cytoglobulin,  nucleohistone. 

ih)  Glycoproteins. — Compounds  of  the  protein  molecule  with  a 
substance  or  substances  containing  a  carbohydrate  group  other  than 
a  nucleic  acid,  e.  g.,  mucins  and  mucoids  {osseomucoid,  tendomucoid, 
ichthulin,  helico protein) . 

(c)  Phosphoproteins. — Compounds  of  the  protein  molecule  with 
some,  as  yet  undefined,  phosphorus-containing  substances  other  than 
a  nucleic  acid  or  lecithin,^  e.  g.,  caseinogen,  vitellin. 

id)  Haemoglobins. — Compounds  of  the  protein  molecule  with 
haematin,  or  some  similar  substance,  e.  g.,  hcemoglohin,  hcemocya- 
nin. 

(e)  Lecithoproteins. — Compounds  of  the  protein  molecule  with 
lecithins,  e.  g.,  lecithans,  phosphatides. 

III.  DERIVED  PROTEINS. 

I.  Primary  Protein  Derivatives. 

Derivatives  of  the  protein  molecule  apparently  formed  through 
hydrolytic  changes  which  involve  only  slight  alteration  of  the  pro- 
tein m.olecule. 

(a)  Proteans. — Insoluble  products  which  apparently  result  from 
the  incipient  action  of  water,  very  dilute  acids  or  enzymes,  e.  g.,  luyo- 
san,  edestan. 

(b)  Metaproteins. — Products  of  the  further  action  of  acids  and 
alkalis  whereby  the  molecule  is  so  far  altered  as  to  form  products 
soluble  in  very  weak  acids  and  alkalis  but  insoluble  in  neutral  fluids, 
e.  g.,  acid  metaprotein  {acid  albuminate),  alkali  metaprotein  {alkali 
albuminate). 

(c)  Coagulated  Proteins. — Insoluble  products  which  result 
from  (i)  the  action  of  heat  on  their  solutions,  or  (2)  the  action  of 
alcohol  on  the  protein. 

*  The  accumulated  chemical  evidence  distinctly  points  to  the  propriety  of  classifying 
the  phosphoproteins  as  conjugated  compounds,  i.  e.,  they  are  possibly  esters  of  some 
phosphoric  acid  or  acids  and  protein. 


86  physiological  chemistry. 

2.  Secondary  Protein  Derivatives.^ 

Products  of  the  further  hydrolytic  cleavage  of  the  protein  molecule. 

(a)  Proteoses. — Soluble  in  water,  non-coagulable  by  heat,  and 
precipitated  by  saturating  their  solutions  with  ammonium — or  zinc 
sulphate,"  e.  g.,  protoproteose,  deutcroproteosc. 

(b)  Peptones. — Soluble  in  water,  non-coagulable  by  heat,  but 
not  precipitated  by  saturating  their  solutions  with  ammonium  sul- 
phate,^ e.  g.,  antipeptone,  am phope plane. 

(c)  Peptides. — Definitely  characterized  combinations  of  two  or 
more  amino  acids,  the  carboxyl  group  of  one  being  united  with  the 
amino  group  of  the  other  with  the  elimination  of  a  molecule  of  water/ 
e.  g.,  dipeptides,  tri peptides,  tetra peptides,  penta peptides. 


CLASSIFICATION  OF  PROTEINS  ADOPTED  BY  THE  CHEM- 
ICAL AND  PHYSIOLOGICAL  SOCIETIES  OF 
ENGLAND. 

I.  Simple  Proteins. 

1.  Protamines,  e.  g.,  sal  mine,  cliipeine. 

2.  Histones,  e.  g.,  globin,  scombrone. 

3.  Albumins,  e.  g.,  ovalbumin,  serum  albumin,  vegetable  albumins. 

4.  Globulins,  e.  g.,  serum  globulin,  ovoglobulin,  vegetable  globulins. 

5.  Glutelins,  e.  g.,  glutenin. 

6.  Alcohol-soluble  proteins,  e.  g.,  zein,  gliadin. 

7.  Scleroproteins,  e.  g.,  elaslin,  keratin. 

8.  Phosphoproteins,  e.  g.,  caseinogen,  vitellin. 


11.  Conju(;atei)  Proteins.. 

1.  (ihi((>|M-olL'ins,  e.  g.,  mucins,  miu:oids. 

2.  Nucleojjroteins,  e.  g.,  nucleohistone,  cyloglobuHn. 

3.  C'hronioproleins,  e.  g.,  hrrmoglobin,  lurmocyanin. 

'  The  term  secondary  protein  dcrivalivcs  is  u.sed  because  tlie  formalion  of  the 
primary  derivatives  usually  precedes  the  formation  of  these  secondary  derivatives. 

'  As  thus  defined,  this  term  does  not  strictly  cover  all  llif  )irot(  in  derivatives  <  (iniinoiily 
called  proteoses,  e.  f^.,  heteroj)roteose  and  dysproteose. 

■'  In  this  )(rouj>  tJie  kyrines  may  be  included.  For  the  present  it  is  believed  tiiat  it  will 
be  helpful  to  retain  this  term  as  defined,  reserving  the  expression  pcjilidc  for  the  simpler 
comjjounds  of  rlefinile  slrucluri-,  such  as  dipeptides,  etc. 

*  'J"he  peptones  are  undoubtedly  peptides  or  ini.xlures  of  peptides,  llir  lallci  Iciin  beiiii^ 
at  jjresent  usefl  to  desij^nate  Iho.sc  of  definite  structure. 


PROTEINS.  87 

III.  Products  of  Protein  Hydrolysis. 

1.  Infraproteins,  e.  g.,  acid  infraprotein  [acid  albuminate),  alkali 
infraprotein  {alkali  albuminate) . 

2.  Proteoses,    e.    g.,    proto proteose,    hetero proteose,    deutero proteose. 

3.  Peptones,  e.  g.,  amphopeptone,  antipeptone. 

4.  Polypeptides,  e.  g.,  di peptides,  tri peptides,  tetrapeptides. 

CONSIDERATIONS  OF  THE  VARIOUS  CLASSES 
OF  PROTEINS. 

SIMPLE  PROTEINS. 

The  simple  proteins  are  true  protein  substances  which,  upon  hy- 
drolysis, yield  only  ct-amino  acids  or  their  derivatives.  "Although 
no  means  are  at  present  a\'ailable  whereby  the  chemical  individu- 
ality of  any  protein  can  be  established,  a  number  of  simple  proteins 
have  been  isolated  from  animal  and  vegetable  tissues  which  have 
been  so  well  characterized  by  constancy  of  ultimate  composition 
and  uniformity  of  physical  properties  that  they  may  be  treated 
as  chemical  individuals  until  further  knowledge  makes  it  possible  to 
characterize,  them  more  definitely."  Under  simple  proteins  we  may 
class  albumins,  globulins,  glutelins,  prolamins,  albuminoids,  his- 
tones  and  protamines. 

ALBUMINS. 

Albumins  constitute  the  first  class  of  simple  proteins  and  may 
be  defined  as  simple  proteins  which  are  coagulable  by  heat  and  soluble 
in  pure  (salt-free)  water.  Those  of  animal  origin  are  not  precipitated 
upon  saturating  their  neutral  solutions  at  30°  C.  with  sodium  chloride 
or  magnesium  sulphate,  but  if  a  saturated  solution  of  this  character 
be  acidified  with  acetic  acid  the  albumin  precipitates.  All  albumins 
of  animal  origin  may  be  precipitated  by  saturating  their  solutions 
with  ammonium  sulpha;te.^  They  may  be  thrown  out  of  solution  by 
the  addition  of  a  sufficient  quantity  of  a  mineral  acid,  whereas  a  weak 
acidity  produces  a  slight  precipitate  which  dissolves  upon  agitating 
the  solution.  Metallic  salts  also  possess  the  property  of  precipitating 
albumins,  some  of  the  precipitates  being  soluble  in  excess  of  the  re- 
agent, whereas  others  are  insoluble  in  such  an  excess.     Of  those  pro- 

^  In  this  connection,  Osborne's  observation  that  there  are  certain  vegetable  albumins 
which  are  precipitated  by  saturating  their  solutions  with  sodium  chloride  or  magnesium 
sulphate  or  by  half-saturating  with  ammonivmi  sulphate,  is  of  interest. 


88  PHYSIOLOGICAL    CHEMISTRY. 

teins  which  occur  native  the  albumins  contain  the  highest  per- 
centage of  sulphur,  ranging  from  1.6  to  2.5  per  cent.  Some  albumins 
have  been  obtained  in  crystalline  form,  notably  egg  albumin,  serum 
albumin,  and  lactalbumin,  but  the  fact  that  they  may  be  obtained 
in  crystalline  form  does  not  necessarily  prove  them  to  be  chemical 
individuals. 

GENERAL  COLOR  REACTIONS  OF  PROTEINS. 

These  color  reactions  are  due  to  a  reaction  between  some  one  or 
more  of  the  constituent  radicals  or  groups  of  the  complex  protein 
molecule  and  the  chemical  reagent  or  reagents  used  in  any  given 
test.  Not  all  proteins  contain  the  same  groups  and  for  this  reason 
the  various  color  tests  will  yield  reactions  \'arying  in  intensity  of  color 
according  to  the  nature  of  the  groups  contained  in  the  particular  pro- 
tein under  examination.  Various  substances  not  proteins  respond  to 
certain  of  these  color  reactions,  and  it  is  therefore  essential  to  submit 
the  material  under  examination  to  several  tests  before  concluding 
definitely  regarding  its  nature. 

TECHNIQUE  OF  THE  COLOR  REACTIONS. 

I.  Millon's  Reaction. — To  5  c.c.  of  a  dilute  solution  of  egg 
albumin  in  a  test-tube  add  a  few  drops  of  Millon's  reagent.  A  white 
precipitate  forms  which  turns  red  when  heated.  This  test  is  a  partic- 
ularly satisfactory  one  for  use  on  solid  proteins,  in  which  case  the 
reagent  is  added  directly  to  the  solid  substance  and  heat  applied, 
which  causes  the  substance  to  assume  a  red  color.  vSuch  proteins 
as  are  not  precipitated  by  mineral  acids,  for  example  certain  of  the 
proteoses  and  peptones,  yield  a  red  solution  instead  of  a  red  precipitate. 

The  reaction  is  due  to  the  presence  of  the  hydroxy -phenyl  group, 
—  C\,H,OH,  in  the  protein  molecule  and  certain  non-j)roteins  such 
as  tyrosine,  phenol  (carbolic  acid)  and  thymol  also  res])ond  to  the 
reaction.  Inasmuch  as  the  tyrosine  grouj)ing  is  the  only  hydroxy- 
[jhenyl  grou{)ing  which  has  definitely  been  proven  to  be  present  in 
the  protein  molecule  it  is  evident  that  protein  substances  respond  to 
.Millon's  reaction  because  of  the  presence  of  this  tyrosine  complex. 
The  test  is  n(;t  a  \ery  satisfactory  one  for  use  in  solutions  containing 
inorganic  .salts  in  large  am(nmt,  since  the  mercury  of  the  Millon's 
reagent*  is  thus  precipitated  and  the  reagent  rendered  inert.     This 

•  Millon's  reagent  consists  of  mercury  dissolved  in  nitric  ik  ifl  loiitainiiif^  some  nitrous 
acid.  It  is  prepared  by  digesting  one  part  (by  weight)  of  iih  n  ury  willi  two  parts  (l)y 
weight)  of  IJNO.,  (sp.  gr.  i  ,42)  and  rljluting  the  resulting  solution  witli  tv\o  volumes  of 
water. 


PROTEINS.  89 

reagent  is  therefore  never  used  for  the  detection  of  protein  material 
in  the  urine. 

2.  Xanthoproteic  Reaction. — To  2-3  c.c.  of  egg  albumin  solu- 
tion in  a  test-tube  add  concentrated  nitric  acid.  A  white  precipi- 
tate forms,  which  upon  heating  turns  yellow  and  finally  dissolves, 
imparting  to  the  solution  a  yellow  color.  Cool  the  solution  and  care- 
fully add  ammonium  hydroxide,  potassium  hydroxide,  or  sodium 
hydroxide  in  excess.  Note  that  the  yellow  color  deepens  into  an 
orange.  This  reaction  is  due  to  the  presence  in  the  protein  molecule 
of  the  phenyl  group,  with  which  the  nitric  acid  forms  certain  nitro 
modifications.  The  particular  complexes  of  the  protein  molecule 
which  are  of  especial  importance  in  this  connection  are  those  of 
tyrosine,  phenylalanine,  and  tryptophane.  The  test  is  not  a  satis- 
factory one  for  use  in  urinary  examination  because  of  the  color  of  the 
end-reaction. 

3.  Adamkiewicz  Reaction. — Thoroughly  mix  i  volume  of  con- 
centrated sulphuric  acid  and  2  volumes  of  acetic  acid  in  a  test-tube, 
add  a  few  drops  of  egg  albumin  solution  and  heat  gently.  A  reddish- 
violet  color  is  produced.  Gelatin  does  not  respond  to  this  test.  This 
reaction  shows  the  presence  of  the  tryptophane  group  (see  next  experi- 
ment). The  test  depends  upon  the  presence  of  glyoxylic  acid,  CHO.- 
COOH-FH^O  or  CH(OH)2COOH,  in  the  reagents.  This  is  shown 
by  the  failure  to  secure  a  positive  reaction  when  acetic  acid  free  from 
glyoxylic  acid  is  used. 

Rosenheim  has  recently  advanced  the  view  that  the  reaction  may 
be  due  to  the  presence  of  oxidizing  agents  such  as  nitrous  acid  and 
ferric  salts  in  the  sulphuric  acid. 

4.  Hopkins-Cole  Reaction.^ — Place  1-2  c.c.  of  egg  albumin  solu- 
'tion  and  3  c.c.  of  glyoxylic  acid,  CHO.COOH  +  H^O  or  CH(0H)2- 

COOH,  solution  (Hopkins-Cole  reagent^)  in  a  test-tube  and  mix 
thoroughly.  In  a  second  tube  place  5  c.c.  of  concentrated  sulphuric 
acid.  Incline  the  tube  containing  the  sulphuric  acid  and  by  means 
of  a  pipette  allow  the  albumin-glyoxylic  acid  solution  to  flow  carefully 
down  the  side.  When  stratified  in  this  manner  a  reddish-violet 
color  forms  at  the  zone  of  contact  of  the  two  fluids.  This  color  is  due 
to  the  presence  of  the  tryptophane  group.  Gelatin  does  not  respond  to 
this  test.     For  formula  for  tryptophane  see  page  72. 

'Hopkins  an-^l  Cole:     Journal  of  Physiology,  XX\^II,  p.  418,  1902. 

*  Hopkins-Cole  reagent  is  prepared  as  follows:  To  one  liter  of  a  saturated  solution 
of  oxalic  acid  add  60  grams  of  sodium  amalgam  and  allow  the  mixture  to  stand  until  the 
evolution  of  gas  ceases.     Filter  and  dilute  with  2-3  volumes  of  water. 


90  PHYSIOLOGICAL    CHEMISTRY. 

Benedict^  has  recently  suggested  a  new  reagent  for  use  in  carrying 
out  the  Hopkins-Cole  reaction. - 

5.  Biuret  Test. — To  2-^  c.c.  of  egg  albumin  solution  in  a  test- 
tube  add  an  equal  volume  of  concentrated  potassium  hydroxide"  solu- 
tion, mix  thoroughly,  and  add  slowly  a  very  dilute  (2-5  drops  in  a 
test-tube  of  water)  cupric  sulphate  solution  until  a  purplish-violet  or 
pinkish-violet  color  is  produced.  The  depth  of  the  color  depends 
upon  the  nature  of  the  protein;  proteoses,  and  peptones  giving  a  de- 
cided pink,  while  the  color  produced  with  gelatin  is  not  far  removed 
from  a  blue.  This  reaction  is  given  by  those  substances  which  con- 
tain lu'o  amino  groups  in  their  molecule,  these  groups  either  being 
joined  directly  together  or  through  a  single  atom  of  nitrogen  or  carbon. 
The  amino  groups  mentioned  must  either  be  two  CONH,  groups 
or  one  COXH,  group  and  one  CSNH,,  C(NH)NH3  or  CH,NH, 
group.  It  follows  from  this  fact  that  substances  which  are  non-protein 
in  character  but  which  contain  the  necessary  groups  will  respond  to 
the  biuret  test.     As  examples  of  such  substances  may  be  cited  oxamide, 

CONH, 

CONH3 

and  biuret, 

CONH2 

\ 
NH. 

/ 
CONH3 

'I'hc  test  derix'cs  its  name  from  the  fact  that  this  latter  substance  which 
is  formed  on  heating  urea  to  180°  C.  (see  page  262),  will  respond  to 
the  test.  Protein  material  responds  positively  since  there  are  two 
CONH2  groups  in  the  protein  molecule. 

According  to  Schiff  the  end-reaction  of  the  biuret  test  is  depend- 
ent upon  the  formation  of  a  co[)per-polassiiin"i  Miirel  compounri  (rupri- 

'  Henc'licl:     Joiinidl  of  Hialoj^ical  Chemistry,  V'l,  p.  51,  lyog. 

^  Uencflicl's  nio'lilicd  HDjjkins-Cole  reagent  is  |jrciiarc(l  as  follows:  Ten  j^ranis  of 
pfjwdered  magnesium  are  jjlaced  in  a  large  Krienmeyer  flask  and  shaken  u])  wiUi  enough  dis- 
tilled water  to  liherally  cover  the  magnesium.  Twohundrtid  and  fifty  (  .(  .of  a  cold,  salurali-d 
solution  of  oxalic  acid  is  now  added  slowly.  The  reaction  prof:eeds  very  rapidly  and  with  the 
liberation  oi  much  heat,  so  that  the  llask  should  he  cooled  under  running  water  during  the 
addition  of  the  acifl.  The  contents  of  the  flask  arc  shaken  after  the  addition  oi  the  last 
portion  of  the  acid  anrl  then  jjoured  ujjon  a  filter,  to  remove  the  insoluble  magnesium 
oxalate.  .A  little  wash  water  is  poured  thrf)Ugh  the  fflter,  the  fill  rate  acidified  with  acetic 
acid  to  prevent  the  partial  precipitation  of  the  magnesium  on  long  standing,  and  made  up 
to  a  liter  with  fjistillefi  water.  This  solution  < onlains  only  the  magncsiuni  sail  of  glyoxylic 
a<if|. 


PROTEINS.  91 

potassium  biuret  or  biuret  potassium  cupric  hydroxide).  This  sub- 
stance was  obtained  by  Schiff  in  the  form  of  long  red  needles.  It 
has  the  following  formula: 

OH  OH 

CO  •  NH2 Cu NH,CO 

\  7 

NH  HN 

/        ■  \ 

CO  •  NH2— K       K— NH2CO 

OH  OH 

Gies^  has  recently  devised  a  reagent  for  use  in  the  biuret  test. 
This  reagent  consists  of  10  per  cent  KOH  solution,  to  which  enough 
3  per  cent  CuSO^  solution  has  been  added  to  impart  a  slight  though 
distinct  blue  color  to  the  clear  hquid.  The  CuSO^  should  be  added 
drop  by  drop  with  thorough  shaking  after  each  addition.  This  reagent 
is  of  material  assistance  in  performing  the  biuret  test. 

6.  Posner's  Modification  of  the  Biuret  Test. — This  test  is  par- 
ticularly satisfactory  for  use  on  dilute  protein  solutions,  and  is  carried 
out  as  follows:  To  some  dilute  egg  albumin  in  a  test-tube  add  one- 
half  its  volume  of  potassium  hydroxide  solution.  Now  hold  the  tube 
in  an  inclined  position  and  allow  some  very  dilute  cupric  sulphate  solu- 
tion, made  as  suggested  on  page  90  (5),  to  flow  down  the  side,  being 
especially  careful  to  prevent  the  fluids  from  mixing.  At  the  juncture 
of  the  two  solutions  the  typical  end-reaction  of  the  biuret  test  should 
appear  as  a  colored  zone  (see  Biuret  Test,  page  90). 

7.  Liebermann's  Reaction. — Add  about  10  drops  of  concen- 
trated egg  albumin  solution  (or  a  little  dry  egg  albumin)  to  about  5  c.c. 
of  concentrated  HCl  in  a  test-tube.  Boil  the  mixture  until  a  pinkish- 
violet  color  results.  This  color  was  originally  supposed  to  indicate 
the  presence  of  a  carbohydrate  group  in  the  protein  molecule,  the  fur- 
furol  formed  through  the  action  of  the  acid  upon  the  protein  reacting 
with  the  hydroxy-phenyl  group  of  the  protein  producing  the  pinkish- 
violet  color.  It  is  now  considered  uncertain  whether  the  carbohydrate 
group  enters  into  the  reaction.  Cole  has  called  attention  to  the  fact 
that  a  blue  color  results  if  protein  material  which  has  been  boiled  with 
alcohol  and  subsequently  washed  with  ether  be  used  in  making  the  test. 
He  believes  the  blue  color  to  be  due  to  an  interaction  between  the 

'  Gies:  Proceedings  of  Society  of  Biological  Chemists,  Journal  of  Biological  Chemistry, 
VII,  p  60,  1910. 


92  PHYSIOLOGICAL    CHEMISTRY. 

i^lyoxylic  acid,  which  was  present  as  an  impurity  in  the  ether  used  in 
washing  the  protein,  and  the  tryptophane  group  of  the  protein  molecule 
which  was  split  off  through  the  action  of  the  acid. 

8.  Acree-Rosenheim  Formaldehyde  Reaction. — Add  a  few 
drops  of  a  dilute  (i  :  5000)  solution  of  formaldehyde  to  2-3  c.c.  of  egg 
albumin  solution  in  a  test-tube.  Mix  thoroughly  and  after  2-3  min- 
utes carefully  introduce  a  little  concentrated  sulphuric  acid  into  the  tube 
in  such  a  manner  that  the  two  solutions  do  not  mix.  A  violet  zone 
will  be  observed  at  the  point  of  juncture  of  the  two  solutions  especially 
if  the  mixture  is  slightly  agitated.  This  color  probably  results  through 
the  union  of  the  protein  and  the  formaldehyde.  If  the  sulphuric  acid 
is  added  to  the  protein  before  the  formaldehyde  is  added  the  typical 
end-reaction  is  not  obtained.  So  far  as  is  known  this  is  a  specific  test 
for  proteins.  The  reaction  cannot  be  applied  satisfactorily  with  con- 
centrated formaldehyde. 

Rosenheim  claims  the  reaction  is  due  to  the  presence  of  oxidizing 
material  in  the  sulphuric  acid  and  that  when  pure  sulphuric  acid  is 
used  no  reaction  is  obtained.  He  advises  the  use  of  a  slight  amount 
of  an  oxidizing  agent,  e.  g.,  ferric  chloride  or  potassium  nitrate  (0.005 
gram  per  100  c.c.  of  sulphuric  acid)  in  order  to  facilitate  the  reaction. 
Rosenheim  further  states  that  proteins  respond  to  the  formaldehyde 
reaction  because  of  the  presence  of  the  tryptophane  group,  a  statement 
which  Acree  does  not  accept  as  proven. 

9.  Bardach's  Reaction.^ — This  is  one  of  the  most  recent  tests 
which  have  been  described  for  the  detection  of  protein  material.  The 
test  depends  upon  the  property  possessed  by  protein  substances  of  pre- 
venting the  formation  of  typical  iodoform  crystals  through  the  inter- 
action of  an  alkaline  acetone  solution  with  iodopotassium  iodide. 
Instead  of  the  typical  hexagonal  plates  or  stellar  formations  of  iodo- 
form there  are  produced,  under  the  conditions  of  the  test,  fine  yellow 
needles  which  are  apparently  some  iodine  compound  other  than  iodo- 
form. The  technique  of  the  test  is  as  follows:  Place  about  5  c.c.  of 
the  protein  solution^  under  examination  in  a  test-tube,  add  2-3  droy)s 
of  a  0.5  per  cent  solution  of  acetone  and  sufficient  Lugol's  .solution"''  to 
supply  a  moderate  excess  of  iodine  and  jjroduce  a  red-brown  coloration. 
(The  amount  of  Lugol's  solution  necessary  will  depend  u])()n  the  con- 
tent of  protein,  sugar,  and  other  iodine-reacting  substances  in  the  solu- 

'  Bardarjh:  ZeUschrift  fiir  Physiologische  Chcmir,  iqoS,  T^rV',  p.  355;  also  Seaman 
and  Gics:  Proceedings  of  the  Society  for  I-.xj)eriment<il.  Jiiolof^y  and  Medicine,  i(;oH,  V,  p.  125  . 

*  The  solution  shrjuWl  not  contain  more  than  5  per  cent  of  jjrolein  material. 

*  Dissolve  4  grams  of  iodine  and  6  grams  of  potassium  iodide  in  100  c.c.  of  distilled 
water. 


PROTEINS.  93 

tion  under  examination  and  may  vary  from  one  drop  to  several  cubic 
centimeters.)  Add  an  excess  (ordinarily  about  3  c.c.j  of  concentrated 
ammonium  hydroxide  and  thoroughly  mix  the  solution.  Place  the  tube 
in  the  test-tube  rack,  examine  the  contents  at  intervals  of  five  minutes, 
and  when  it  is  evident  that  crystals  have  formed,  place  a  drop  of  the 
mixture  upon  a  microscopic  slide,  put  a  coverglass  in  position,  and 
examine  the  mixture  under  the  microscope.  The  formation  of  canary 
yellow  crystals  indicates  the  presence  of  protein  material  in  the  solution 
examined.  The  crystals  are  ordinarily  needle-like  in  appearance  and 
show  a  tendency  to  assume  rosette  or  bundle-like  formations,  but  under 
certain  conditions  they  may  show  knobbed  (nail-like)  and  branching 
variations. 

If  a  moderate  excess  of  iodine  is  used  in  making  the  test,  a  black 
precipitate  of  iodonitro  compounds  is  at  once  formed  upon  the  addition 
of  the  ammonium  hydroxide,  and  yellow  needles  are  subsequently 
deposited  upon  it.  In  case  just  the  proper  amount  of  iodine  is  used, 
the  solution  soon  assumes  a  yellow  color  and  the  black  precipitate 
formed  upon  the  addition  of  the  ammonium  hydroxide  is  gradually 
transformed  more  or  less  completely  into  the  yellow  crystals.  In  either 
case  the  needles  ordinarily  form  within  an  hour,  and  frequently  in  a 
much  shorter  time.  If  too  great  an  excess  of  iodine  is  employed  the 
heavy  black  precipitate  may  obscure  or  even  prevent  the  reaction. 
The  presence  of  insufficient  iodine  or  excess  protein  may  likewise  pre- 
vent the  reaction.  In  tests. in  which  a  concentrated  protein  solution 
and  an  excess  of  iodine  are  used,  the  addition  of  ammonium  hydroxide 
immediately  produces  a  grayish-green  precipitate.  In  such  instances, 
if  the  proportions  are  favorable,  and  the  mixture  be  stirred  with  a 
glass  rod  for  a  few  minutes,  the  precipitate  is  gradually  transformed 
into  the  crystals  before  mentioned. 

It  is  probable  that  all  soluble  proteins  will  respond  to  Bardach's 
reaction,  but  the  relative  delicacy  of  the  reaction  as  well  as  the  value 
of  the  test  as  compared  with  other  protein  tests  remain  to  be  determined. 
The  only  disturbing  factor  noted  thus  far  is  the  presence  of  earthy 
phosphates  in  the  solution  under  examination. 

PRECIPITATION  REACTIONS  AND  OTHER  PROTEIN  TESTS. 

There  are  three  forms  in  which  proteins  may  be  precipitated,  i.  e., 
unaltered,  as  an  albuminate,  and  as  an  insoluble  salt.  An  instance  of 
the  precipitation  in  a  native  or  unaltered  condition  is  seen  in  the  so- 
called   salting-out   experiments.     Various   salts,   notably    (NHJgSO^, 


94  PHYSIOLOGICAL    CHEMISTRY. 

ZnSO^.  MgSO^,  XajSO^  and  NaCl  possess  the  power  when  added  in 
solid  form  to  certain  definite  protein  solutions,  of  rendering  the  men- 
struum incapable  of  holding  the  protein  in  solution,  thereby  causing 
the  protein  to  be  precipitated  or  salted-out  to  use  the  common  term. 
Mineral  acids  and  alcohol  also  precipitate  proteins  unaltered.  Pro- 
teins are  precipitated  as  albuminates  when  treated  with  certain  metallic 
salts,  and  precipitated  as  insoluble  salts  when  weak  organic  acids  such 
as  certain  of  the  alkaloidal  reagents  are  added  to  their  solutions. 

It  is  generally  stated  that  globulins  are  precipitated  from  their 
solutions  upon  Jialf  saturation  with  ammonium  sulphate  and  that 
albumins  are  precipitated  upon  complete  saturation  by  this  salt.  Com- 
paratively few  exceptions  were  found  to  this  rule  until  proteins  of 
vegetable  origin  came  to  be  more  extensively  studied.  These  studies, 
furthered  especially  by  Osborne  and  associates,  have  demonstrated 
\ery  clearly  that  the  characterization  of  a  globulin  as  a  protein  which 
is  precipitated  by  half  saturation  with  ammonium  sulphate,  can  no 
longer  hold.  Certain  vegetable  globulins  have  been  isolated  which 
are  not  precipitated  by  this  salt  until  a  concentration  is  reached  greater 
than  that  secured  by  half-saturation.  As  an  example  of  an  albumin 
which  does  not  conform  to  the  definition  of  an  albumin  as  regards  its 
precipitation  by  ammonium  sulphate,  may  be  mentioned  the  leucosin 
of  the  wheat  germ  which  is  precipitated  from  its  solution  upon  half- 
saturation  with  ammonium  sulphate.  The  limits  of  precipitation  by 
ammonium  sulphate,  therefore,  do  not  furnish  a  suf^ciently  accurate 
basis  for  the  differentiation  of  globulins  from  albumins.  It  has  further 
been  determined  that  a  given  protein  which  is  precipitablc  by  ammo- 
nium sulphate  cannot  be  ''salted-out"  by  the  same  concentration  of 
the  sah  under  all  ronditions. 

EXPKRIMENTS. 

I.  Influence  of  Concentrated  Mineral  Acids,  Alkalis  and 
Organic  Acids.  Prejnire  fi\e  test  tubes  each  containing  5  c.c.  of 
c(;ncentrated  egg  albumin  solution.  'Yo  the  first  add  concentrated 
HjSO,,  (lro|)  \)y  clrop,  uniil  an  excess  of  the  acid  has  been  added. 
Xote  any  changes  which  may  occur  in  the  solution.  Alhjw  the  tube 
to  stand  for  24  hours  and  at  the  end  of  that  ])eriod  obserxe  any  altera- 
tion which  may  ha\'e  taken  |)lace.  Heal  the  lul)caiid  note  any  further 
change  which  may  occur.  Repeat  the  experiment  in  the  four  remain- 
ing lubes  with  concentrated  hydrochloric  acid,  concentrated  nitric 
acid,  concentrated  jxjtassium    hydroxide  and  acetic  acid.     How  do 


PROTEINS.  95 

Strong  mineral  acids,  strong  alkalis,  and  strong  organic  acids  differ  in 
their  action  toward  protein  solutions  ? 

2.  Precipitation  by  Metallic  Salts. — Prepare  four  tubes  each  con- 
taining 2-^  c.c.  of  dilute  egg  albumin  solution.  To  the  first  add  mer- 
curie  chloride,  drop  by  drop,  until  an  excess  of  the  reagent  has  been 
added,  noting  any  changes  which  may  occur.  Repeat  the  experiment 
with  plumbic  acetate,  argentic  nitrate,  cupric  sulphate,  ferric  chloride, 
and  barium  chloride. 

Egg  albumin  is  used  as  an  antidote  for  lead  or  mercury  poisoning. 
Why? 

3.  Precipitation  by  Alkaloidal  Reagents. — Prepare  six  tubes 
each  containing  2-3  c.c.  of  dilute  egg  albumin  solution.  To  the  first 
add  picric  acid  drop  by  drop  until  an  excess  of  the  reagent  has  been 
added,  noting  any  changes  which  may  occur.  Repeat  the  experiment 
with  trichloracetic  acid,  tannic  acid,  phosphotungstic  acid,  phospho-molyb- 
dic  acid,  and  potassio-mercuric  iodide.  Acidify  with  hydrochloric  acid 
before  testing  with  the  three  last  reagents. 

4.  Heller's  Ring  Test. — Place  5  c.c.  of  concentrated  nitric  acid 
in  a  test-tube,  incline  the  tube,  and  by  means  of  a  pipette  allow  the 
dilute  albumin  solution  to  flow  slowly  down  the  side.  The  liquids 
should  stratify  with  the  formation  of  a  white  zone  of  precipitated 
albumin  at  the  point  of  juncture.  This  is  a  very  delicate  test  and  is 
further  discussed  on  p.  308. 

An  apparatus  called  the  albumoscope  or  horismascope  has  been 
devised  for  use  in  the  tests  of  this  character  and  has  met  with  consider- 
able favor.     The  method  of  using  the  albumoscope  is  described  below. 

Use  of  the  Albumoscope. — This  instrument  is  intended  to  facilitate 
the  making  of  "ring"  tests  such  as  Heller's  and  Roberts'.  In  making 
a  test  about  5  c.c.  of  the  solution  under  examination  is  first  introduced 
into  the  apparatus  through  the  larger  arm  and  the  reagent  used  in  the 
particular  test  is  then  introduced  through  the  capillary  arm  and  allowed 
to  flow  down  underneath  the  solution  under  examination.  If  a  reason- 
able amount  of  care  is  taken  there  is  no  possibility  of  mixing  the  two 
solutions  and  a  definitely  defined  white  "ring"  is  easily  obtained  at 
the  zone  of  contact. 

5.  Roberts'  Ring  Test. — Place  5  c.c.  of  Roberts'  reagent^  in  a 
test-tube,  incline  the  tube,  and  by  means  of  a  pipette  allow  the  al- 
bumin solution  to  flow  slowly  down  the  side.  The  liquids  should 
stratify  with  the  formation  of  a  ivhite  zone  of  precipitated  albumin  at 

^  Roberts'  reagent  is  composed  of  i  volume  of  concentrated  HNO)  and  5  volumes 
of  a  saturated  solution  of  MgSO^. 


96  PHYSIOLOGICAL    CHEMISTRY. 

the  point  of  juncture.  This  test  is  a  modification  of  Heller's  ring 
test  and  is  rather  more  satisfactory.  The  albumoscope  may  also  be 
used  in  making  this  test.     (See  page  95.) 

6.  Spiegler's  Ring  Test. — Place  5  c.c.  of  Spiegler's  reagent^ 
in  a  test-tube,  inch'ne  the  tube,  and  by  means  of  a  pipette  allow  5  c.c. 
of  albumin  solution,  acidified  with  acetic  acid,  to  flow  slowly  down  the 
side.  A  white  zone  will  form  at  the  point  of  contact.  This  is  an 
exceedingly  delicate  test,  in  fact  too  delicate  for  ordinary  clinical  pur- 
poses, since  it  serves  to  detect  albumin  when  present  in  the  merest 
trace  (1:250,000).     This  test  is  further  discussed  on  page  310. 

7.  Jolles'  Reaction. — Shake  5  c.c.  of  albumin  solution  with  i 
c.c.  of  30  per  cent  acetic  acid  and  4  c.c.  of  Jolles'  reagent"  in  a  test-tube. 
A  white  precipitate  of  albumin  should  form.  Care  should  be  taken  to 
use  the  correct  amount  of  acetic  acid.  For  further  discussion  of  the 
test  see  page  310. 

8.  Tanret's  Test. — To  5  c.c.  of  albumin  solution  in  a  test-tube 
add  Tanret's  reagent,"''  drop  by  droj),  until  a  turl^idity  or  precipitate 
forms.  This  is  an  exceedingly  delicate  test.  Sometimes  the  albumin 
solution  is  stratified  upon  the  reagent  as  in  Heller's  or  Roberts'  ring 
tests.  In  urine  examination  it  is  claimed  by  Repiton  that  the  presence 
of  urates  lowers  the  delicacy  of  the  test.  Tanret  has,  however,  very 
recently  made  a  statement  to  the  effect  that  the  remo\al  of  urates  is 
not  necessary  inasmuch  as  the  urate  precipitate  will  disappear  on 
warming  and  the  all)umin  ])re(i])ilatc  will  not.  He  says,  however, 
that  mucin  interferes  with  the  delicacy  of  his  test  and  should  be  removed 
by  acidification  with  acetic  acid  and  filtration  before  testing  for  albumin. 

9.  Sodium  Chloride  and  Acetic  Acid  Test.  Mix  2  volumes 
of  albumin  sfjlution  and  1  xohime  of  a  saturated  solution  of  sodium 
chloride  in  a  test-tube,  acidify  with  acetic  acid,  and  heat  to  boiling. 

The  production  of  a  cloudiness  or  the  formation  of  a  precipitate  indi- 
cates the  presence  of  albumin. 

'  S[)iegler's  reagent  has  Uie  foli<n\ing  K^nipo.sition: 

'I'artaric  acid    jo  giani.s. 

.Mercuric  chlorirle 40  grams. 

(ilyccrol 100  grains. 

Distilled  water  icco  grams. 

-  Jolles'  reagent  has  the  following  composition: 

Succinic  jicid \o  grams. 

Mercuric  chloride    20  grams. 

Sodium  chloride    20  grams. 

Distilled  water 1 000  grams. 

^Tanret's  reagent  is  prejjarcd  as  follows:  Dissolve  1.35  gram  of  men  uric  chloride 
in  25  c.c.  of  water,  add  to  this  soliiliori  -^ .  -52  grams  of  j)otassiimi  iodide  dissolved  in  25  c.c. 
of  water,  then  malce  the  tola!  solution  u|;  to  (m  1  j  .  v.iili  w.iii  1  :iiid  .idd  20  c.c.  of  glacial 
acetic  arid  to  tlie  coiirMncd  solulions. 


PROTEINS.  97 

10.  Potassium  Iodide  Test. — Stratify  a  dilute  albumin  solution 
upon  a  solution  of  potassium  iodide  made  slightly  acid  with  acetic  acid. 
In  the  presence  of  0.01-0.02  per  cent  of  albumin  a  white  ring  forms 
immediately.  If  the  test  be  allowed  to  stand  two  minutes  after  the 
stratification  it  will  serve  to  detect  0.005  P^r  cent  of  albumin. 

11.  Acetic  Acid  and  Potassium  Ferrocyanide  Test. — To  5  c.c. 
of  dilute  egg  albumin  solution  in  a  test-tube  add  5-10  drops  of  acetic 
acid.  Mix  well,  and  add  potassium  ferrocyanide,  drop  by  drop,  until 
a  precipitate  forms.     This  test  is  very  delicate. 

Schmiedl  claims  that  a  precipitate  of  Fe(Cn)gK2Zn  or  Fe(Cn)g- 
Zng,  is  formed  when  solutions  containing  zinc  are  subjected  to  this 
test,  and  that  this  precipitate  resembles  the  precipitate  secured  with 
protein  solutions.  In  the  case  of  human  urine  a  reaction  was  obtained 
when  0.000022  gram  of  zinc  per  cubic  centimeter  was  present. 
Schmiedl  further  found  that  the  urine  collected  from  rabbits  housed  in 
zinc-lined  cages  possessed  a  zinc  content  which  was  sufficient  to 
yield  a  ready  response  to  the  test.  Zinc  is  the  only  interfering 
substance  so  far  reported. 

12.  Salting-out  Experiments. — {a)  To  2^,  c.c.  of  egg  albumin 
solution  in  a  small  beaker  add  solid  ammonium  sulphate  to  the  point 
of  saturation,  keeping  the  temperature  of  the  solution  below  40°  C. 
Filter,  test  the  precipitate  by  Millon's  reaction  and  the  filtrate  by  the  biu- 
ret test.  What  are  your  conclusions?  (h)  Repeat  the  above  experi- 
ment making  the  saturation  with  solid  sodium  chloride.  How  does 
this  result  differ  from  the  result  of  the  saturation  with  ammonium  sul- 
phate? Add  2-3  drops  of  acetic  acid.  What  occurs?  All  proteins 
except  peptones  are  precipitated  by  saturating  their  solutions  with  ammo- 
nium sulphate.  Globulins  are  the  only  proteins  precipitated  by  satu- 
rating with  sodium  chloride  (see  Globulins,  page  100),  unless  the  satu- 
rated solution  is  subsequently  acidified,  in  which  event  all  proteins 
except  peptones  are  precipitated. 

Soaps  may  be  salted-out  in  a  similar  manner  (see  p.  134). 

13.  Coagulation  or  Boiling  Test. — Heat  25  c.c.  of  dilute  egg 
albumin  solution  to  the  boiling-point  in  a  small  evaporating  dish. 
Xhe  albumin  coagulates.  Complete  coagulation  may  be  obtained  by 
acidifying  the  solution  with  3-5  drops  of  acetic  acid^  at  the  boiling- 
point.  Test  the  coagulum  by  Millon's  reaction.  The  acid  is  added 
to  neutralize  any  possible  alkalinity  of  the  solution,  and  to  dissolve  any 
substances  which  are  not  albumin  (see  further  discussion  on  page  311). 

^  Nitric  acid  is  often  used  in  place  of  acetic  acid  in  this  test.  In  case  nitric  acid  is 
used,  ordinarily  1-2  drops  is  sufficient. 


98 


PHYSIOLOGICAL    CHEMISTRY. 


OB 


14.  Coagulation  Temperature. — Prepare  4  test-tubes  each  con- 
taining 5  c.c.  of  neutral  egg  albumin  solution.  To  the  first  add  i  drop 
of  0.2  per  cent  hydrochloric  acid,  to  the  second  add  i  drop  of  0.5  per 
cent  sodium  carbonate  solution,  to  the  third  add  i  drop  of  10  per  cent 
sodium  chloride  solution   and   leave  the  fourth  neutral   in  reaction. 

Partly  fill  a  beaker  of  medium  size  with 
water  and  place  it  within  a  second 
larger  beaker  which  also  contains  water, 
the  two  vessels  being  separated  by 
pieces  of  cork.  Fasten  the  four  test- 
tubes  compactly  together  by  means  of  a 
rubber  band,  lower  them  into  the  water 
of  the  inner  beaker  and  suspend  them, 
by  means  of  a  clamp  attached  to  one  of 
the  tubes,  in  such  a  manner  that  the 
albumin  solutions  shall  be  midway  be- 
tween the  upper  and  lower  surfaces  of 
the  water.  In  one  of  the  tubes  place 
a  thermometer  with  its  balb  entirely 
beneath  the  surface  of  the  albumin 
solution  (Fig.  32).  Gently  heat  the 
water  in  the  beakers,  noting  carefully 
any  changes  which  may  occur  in  the 
albumin  solutions  and  record  the  exact 
temperature  at  which  these  changes 
occur.  The.  first  appearance  of  an 
opacity  in  an  albumin  solution  indicates 
the  commencement  of  coagulation  and 
the  temperature  at  which  this  occurs 
should  be  recorded  as  the  coagulation 
temperature  for  that  ])arlicular  albumin 
solution. 

What  is  the  order  in  which  the  four  solutions  coagulate? 
Repeat  the  experiment,  adding  to  the  first  tube  1   drop  of  acetic 
acid,  to  the  second   i  dr(;p  of  concentrated  potassium  hydroxide  solu- 
tion, to  the  third  2  drops  of  a  10  jier  cent  sodium  (  hloridc  solution  and 
leave  the  fourth  neutral  as  before. 

What  is  the  order  of  coagulation  here?     Why? 

15.  Precipitation   by  Alcohol.     Pre})are  .3   test-tubes  each  < on 
taining  about  10  c.  c.  of  95  per  cent  alcohol.     To  the  first  add  one  droj) 
of  0.2  per  cent  hydrochloric  acid,  to  the  second  one  drop  of  potassium 


Ik 


32.— Coagulation  Tkmi'kk - 
ATUKE  Apparatus. 


PROTEINS.  99 

hydroxide  solution  and  leave  the  third  neutral  in  reaction;  Add  to 
each  tube  a  few  drops  of  egg  albumin  solution  and  note  the  results. 
What  do  you  conclude  from  this  experiment  ?  Alcohol  precipitates  pro- 
teins unaltered,  but  if  allowed  to  remain  under  alcohol  the  protein  is 
transformed.  The  "fixing"  of  tissues  for  histological  examination  by 
means  of  alcohol  is  an  illustration  of  the  application  of  this  trans- 
formation produced  by  alcohol.  It  apparently  is  a  process  of 
dehydrolysis. 

i6.  Preparation  of  Powdered  Egg  Albumin. — This  may  be 
prepared  as  follows:  Ordinary  egg-white  finely  divided  by  means  of 
scissors  or  a  beater  is  treated  with  four  volumes  of  water  and  filtered. 
The  filtrate  is  evaporated  on  a  water-bath  at  about  50°  C.  and  the 
residue  powdered  in  a  mortar. 

17.  Tests  on  Powdered  Egg  Albumin. — With  powdered  albu- 
min prepared  as  described  above  (by  yourself  or  furnished  by  the 
instructor),  try  the  following  tests: 

(a)  Solubility. 

(b)  MiUon's  Reaction,. 

{c)  Hopkins-Cole  Reaction. — When  used  to  detect  the  presence 
of  protein  in  solid  form  this  reaction  should  be  conducted  as  follows: 
Place  5  c.c.  of  concentrated  sulphuric  acid  in  a  test-tube  and  add  care- 
fully, by  means  of  a  pipette,  3-5  c.c.  of  Hopkins-Cole  reagent.  Intro- 
duce a  small  amount  of  the  solid  substance  to  be  tested,  agitate  the  tube 
slightly,  and  note  that  the  suspended  pieces  assume  a  reddish-violet 
color,  which  is  the  characteristic  end-reaction  of  the  Hopkins-Cole 
test;  later  the  solution  will  also  assume  the  reddish- violet  color. 

{d)  Composition  Test. — Heat  some  of  the  powder  in  a  test-tube 
in  which  is  suspended  a  strip  of  moistened  red  litmus  paper  and  across 
the  mouth  of  which  is  placed  a  piece  of  filter  paper  moistened  with 
plumbic  aceate  solution.  As  the  powder  is  heated  it  chars,  indicating 
the  presence  of  carbon;  the  fumes  of  ammonia  are  evolved,  turning 
the  red  litmus  paper  blue  and  indicating  the  presence  of  nitrogen  and 
hydrogen;  the  plumbic  acetate  paper  is  blackened,  indicating  the  pres- 
ence of  sulphur,  and  the  deposition  of  moisture  on  the  side  of  the  tube 
indicates  the  presence  of  hydrogen. 

(e)  Immerse  a  dry  test-tube  containing  a  little  powdered  egg 
albumin  in  boihng  water  for  a  few  moments.  Remove  and  test  the 
solubility  of  the  albumin  according  to  the  directions  given  under  (a) 
above.  It  is  still  soluble.  Why  has  it  not  been  coagulated  ?  Repeat 
the  above  experiments  with  powdered  serum  albumin  and  see  how  the 
results  compare  with  those  just  obtained. 


lOO  PHYSIOLOGICAL    CHEMISTRY. 

SULPHUR  IX  PROTEIN. 

Sulphur  is  belie\cd  to  be  present  in  two  different  forms  in  the  pro- 
tein molecule.  The  first  form,  which  is  present  in  greatest  amount, 
is  that  loosely  combined  with  carbon  and  hydrogen.  Sulphur  in  this 
form  is  variously  termed  iinoxidized,  loosely  combined,  mercaptan,  and 
lead-blackening  sulphur.  The  second  form  is  combined  in  a  more 
stable  manner  with  carbon  and  oxygen  and  is  known  as  oxidized  or 
acid  sulphur.  The  protamines  are  the  only  class  of  sulphur-free 
proteins. 

Tests  for  Sulphur. 

1.  Test  for  Loosely  Combined  Sulphur. — To  ec^ual  volumes  of 
KOH  and  egg  albumin  solutions  in  a  test-tube  add  1-2  drops  of  plumbic 
acetate  solution  and  boil  the  mixture.  Loosely  combined  sulphur  is 
indicated  by  a  darkening  of  the  solution,  the  color  deepening  into  a 
black  if  sufficient  sulphur  is  present.  Add  hydrochloric  acid  and  note 
the  characteristic  odor  evohed  from  the  solution.  Write  the  reactions 
for  this  test. 

2.  Test  for  Total  Sulphur  (Loosely  Combined  and  Oxidized). 
— Place  the  substance  to  be  examined  (powdered  egg  albumin)  in  a 
small  procclain  crucible,  add  a  suitable  amount  of  solid  fusion  mixture 
(potassium  hydroxide  and  potassium  nitrate  mixed  in  the  proportion 
5:1)  and  heat  carefully  until  a  colorless  mixture  results.  (Sodium  per- 
oxide may  be  used  in  place  of  this  fusion  mixture  if  desired.)  Cool, 
dissolve  the  cake  in  a  little  warm  water  and  filter.  Acidify  the  filtrate 
with  hydrochloric  acid,  heat  it  to  the  boiling-point  and  add  a  small 
amount  (;f  barium  chloride  solution.  A  white  preci])itate  forms  if 
sulphur  is  })resent.     What  is  this  prccij)itate? 

GLOBULINS. 

Globulins  arc  simple  ]jroteins  especially  predominant  in  the  vege- 
table kingdom.  They  are  closely  related  to  the  albumins  and  in  com- 
mon with  them  gi\e  all  the  ordinary  protein  tests,  (llobulins  differ 
from  the  albumins  in  being  insoluljlc  in  |)ure  fsah  free)  water.  They 
are,  however.  solui)le  in  neutral  s(;lutions  of  salts  of  strong  ba.ses  with 
strong  acids.  Most  globulins  are  precij.)itated  from  their  solutions  by 
saturation  with  solid  sodium  chloride  or  magnesium  sulphate.  As  a 
class  they  are  much  less  stable  than  the  albumins,  a  fad  shown  by  the 
increasing  dilTiculty  with  which  a  gloljiiliii  dissoKcs  during  the  course 
of  successive  repreci])italions. 


PROTEINS. 


lOI 


We  have  used  an  albumin  of  animal  origin  (egg  albumin)  for  all 
the  protein  tests  thus  far,  whereas  the  globulin  to  be  studied  will  be 
prepared  from  a  vegetable  source.  There  being  no  essential  difference 
between  animal  and  vegetable  proteins,  the  vegetable  globulin  we  shall 
study  may  be  taken  as  a  true  type  of  all  globulins,  both  animal  and 
vegetable. 

Experiments  on  Globulin. 

Preparation  of  the  Globulin. — Extract  20-30  grams  (a  handful) 
of  crushed  hemp  seed  with  a  5  per  cent  solution  of  sodium  chloride 
for  one-half  hour  at  60°  C.  Filter  while  hot  through  a  paper  moistened 
with  5  per  cent  sodium  chloride  solution.  Place  the  filtrate  in  the 
water-bath  at  60°  C.  and  allow  it  to  stand  for  24  hours  in  order  that 
the  globulin  may  crystallize  slowly.     In  case  the  filtrate  is  clotidy  it 


Fig.  t,t,. — Edestin. 


should  be  warmed  to  60°  C.  in  order  to  produce  a  clear  solution.  The 
globulin  is  soluble  in  hoi  5  per  cent  sodium  chloride  solution  and  is  thus 
extracted  from  the  hemp  seed,  but  upon  cooling  this  solution  much  of 
the  globulin  separates  in  crystalline  form.  This  particular  globulin 
is  called  edestin.  It  crystallizes  in  several  different  forms,  chiefly 
octahedra  (see  Fig.  t,t,,  above).  (The  crystalline  form  of  excelsm, 
a  protein  obtained  from  the  Brazil  nut,  is  shown  in  Fig.  34,  page 
103.  This  vegetable  protein  crystallizes  in  the  form  of  hexagonal 
plates.)  Filter  off  the  edestin  and  make  the  following  tests  on  the 
crystalline  body  and  on  the  filtrate  which  still  contains  some  of  the 
extracted  tjlobulin. 


I02  PHYSIOLOGICAL    CHEMISTRY. 

Tests  on  Crystallized  Edestix. — (i)  Microscophal  examwa- 
tian  (see  Fig.  t,t,,  p.  loi). 

(2)  Solubility. — Try  the  solubility  in  the  ordinary  sohents  (see 
page  22).  Keep  these  solubilities  in  mind  for  comparison  with  those 
of  edestan,  to  be  made  later  (see  page  107). 

(3)  Millons  Reaction. 

(4)  Coagulation  7"<?5/.— Place  a  small  amount  of  the  globulin  in  a 
test-tube,  add  a  little  water  and  boil.  Now  add  dilute  hydrochloric 
acid  and  note  that  the  protein  no  longer  dissolves.  It  has  been 
coagulated. 

(5)  Dissolve  the  remainder  of  the  edestin  in  0.2  per  cent  hydro- 
chloric acid  and  preserve  this  acid  solution  for  use  in  the  experiments 
on  proteans  (see  page  106). 

Tests  on  Edestin  Filtrate. — (i)  Influence  of  Protein  Precipi- 
tants. — Try  a  few  protein  precipitants  such  as  nitric  acid,  tannic  acid, 
picric  acid,  and  mercuric  chloride. 

(2)  Biuret  Test. 

(3)  Coagulation  Test. — Boil  some  of  the  filtrate  in  a  test-tube. 
What  happens  ? 

(4)  Saturation  with  Sodium  Chloride. — Saturate  some  of  the  fil- 
trate with  solid  sodium  chloride.  How  does  this  result  differ  from  that 
obtained  upon  saturating  egg  albumin  solution  with  solid  sodium 
chloride  ? 

(5)  Precipitation  by  Dilution. — Dilute  some  of  the  filtrate  with 
ia-15  volumes  of  water.     Why  does  the  globulin  precipitate? 

Glutelins. 

It  has  been  repeatedly  shown,  particularly  by  ( )sborne,  that  after 
extracting  the  seeds  of  cereals  with  water,  neutral  salt  solution,  and 
strong  alcohol,  there  still  remains  a  residue  which  contains  ])rotein 
material  which  may  Ije  extracted  by  \ery  dilute  acid  or  alkali.  These 
proteins  which  are  insoluble  in  all  neutral  soKents,  but  readily  soluble 
in  very  dilute  acids  and  alkalis  are  called  glutelins.  The  only  member 
of  the  gnjup  which  has  yet  received  a  name,  is  the  glutenin  of  wheat, 
a  protein  which  constitutes  nearly  50  per  cent  of  the  gluten.  It  is 
not  defmitely  known  whether  glutelins  occur  as  constituents  of  all  seeds. 

Prolamins  (Alcohol-soluble  Proteins). 

The  term  prolamin  has  been  jjroposed  by  Osborne,  for  the  grouj) 
of  j)roteins   ff)rmerly   Icrmcrl    "alcohol  soluble   proteins.'"     The   name 


PROTEINS. 


103 


is  very  appropriate  inasmuch  as  these  proteins  yield,  upon  hydrolysis, 
especially  large  amounts  of  proline  and  ammonia.  The  prolamins  are 
simple  proteins  which  are  insoluble  in  water,  absolute  alcohol  and  other 
neutral  solvents,  but  are  soluble  in  70  to  80  per  cent  alcohol  and  in 
dilute  acids  and  alkalis.  They  occur  widely  distributed,  particularly 
in  the  vegetable  kingdom.  The  only  prolamins  yet  described  are  the 
zein  of  maize,  the  hordein  of  barley,  the  gliadin  of  wheat  and  rye,  and 
the  hynin  of  malt.     They  yield  relatively  large  amounts  of  glutamic 


Fig.  j4. — ExcELsiN,  the  Protein  of  the  Br.azil  Nut. 
(Drawn  from  crystals  furnished  by  Dr.  Thomas  B.  Osborne,  New  Haven,  Conn.) 

acid  on  hydrolysis  but  no  lysin.  The  largest  percentage  of  glutamic 
acid  (41.32  per  cent)  ever  obtained  as  a  decomposition  product 
of  a  protein  substance  has  very  recently  been  obtained  by  Klein- 
schmitt  from  the  hydrolysis  of  the  prolamin  hordein.^  This  yield 
of  glutamic  acid  is  also  the  largest  amount  of  any  single  decomposition 
product  yet  obtained  from  any  protein  except  protamines. 


Albuminoids.     (Scleroproteins.) 

The  albuminoids  yield  similar  hydrolytic  products  to  those  obtained 
from  the  other  simple  proteins  already  considered,  thus  indicating  that 
they  possess  essentially  the  same  chemical  structure.  They  differ 
from  all  other  proteins,  whether  simple,  conjugated,  or  derived,  in  that 
they  are  insoluble  in  all  neutral  solvents.  The  albuminoids  include 
"the  principal  organic  constituents  of  the  skeletal  structure  of  animals 

*  Up  to  this  time  the  yield  of  37.33  per  cent  obtained  by  Osborne  and  Harris  from 
gliadin,  was  the  maximum  yield. 


I04  PHYSIOLOGICAL    CHEMISTRY. 

as  well  as  their  external  covering  and  its  appendages.  Some  of  the 
principal  albuminoids  are  keratin,  elastin,  collagen,  reticulin,  spongin, 
and  fibroin.  Gelatin  cannot  be  classed  as  an  albuminoid  although  it 
is  a  transformation  product  of  collagen.  The  various  albuminoids 
differ  from  each  other  in  certain  fundamental  characteristics  which 
will  be  considered  in  detail  under  Epithelial  and  Connective  Tissue 
(see  Chapter  XIV,  p.  223). 

CONJUGATED  PROTEINS. 

Conjugated  proteins  consist  of  a  protein  molecule  united  to  some 
other  molecule  or  molecules  otherwise  than  as  a  salt.  .  We  have  glyco- 
proteins, nucleo proteins,  hcemoglobins  (chromoproteins),  phosphopro- 
teins  and  lecithoproteins  as  the  five  classes  of  conjugated  proteins. 

Glycoproteins  may  be  considered  as  compounds  of  the  protein 
molecule  with  a  substance  or  substances  containing  a  carbohydrate 
group  other  than  a  nucleic  acid.  The  glycoproteins  yield,  upon  decom- 
position, protein  and  carbohydrate  derivatives,  notably  glycosamine, 
CH,OH.(CHOH)3.CH(NH,).CHO,  and  galactosamine,  OHCH^.- 
(CHOH)3.CH(NH2).CHO.  The  principal  glycoproteins  are  mucoids, 
mucins,  and  chondro proteins.  By  the  term  mucoid  we  may  designate 
those  glycoproteins  which  occur  in  tissues,  such  as  tendomucoid  from 
tendinous  tissue  and  osseomucoid  from  l)one.  The  elementary  com- 
[josition  of  these  typical  mucoids  is  as  follows: 

N.  S.  C.         H.  0 

'J'endomucoid    ii-75      --ii      4S.76      6.5:;      .^0.60  (('hillLTiden  and  Gies) 

Osseomucoid       12.22      2.32      47-4,?      ^>-63      31-40 

The  term  mucins  may  be  said  to  include  those  forms  of  glycoproteins 
which  occur  in  the  secretions  and  fluids  of  the  l)ody.  Chondropro- 
teins  are  so  named  because  cliondromucoid,  the  })rincij)al  member  of 
the  gr<jup,  is  derived  from  cartilage  (chondrigen).  Amyloid,  which 
appears  ])athologically  in  the  spleen,  li\er,  and  kidneys,  is  also  a 
chondroprotein. 

The  nwieoproteins  occur  |)rincipally  in  animal  and  \egelable  cells, 
and  following  the  destruction  of  these  (ells  they  are  found  in  the  fluids 
of  the  body.  'I'hese  proteins  are  discharged  into  the  tissue  fluids  by 
the  activity  or  disintegration  of  cells.  Combined  with  the  sim])le  pro- 
tein in  the  neucleoprotein  molecule  we  find  nucleic  acid,  a  body  which 
contains  phosphorus  and  which  yields  purine  bases  and  pyrimidine 
bases  {thymine,  cytosine,  and  uracil)  upon  decomposition.  The  so-called 
nucleins  are  formed  in  the  gastric  digestion  of  nucleoprolcins. 


PROTEINS.  105 

Wheeler-Johnson  Reaction  for  Uracil  and  Cytosine. — To  about 
5  c.c.  of  the  sohition  under  examination  add  bromine  water  until  the 
color  is  permanent/  In  case  the  solution  contains  only  small  quan- 
tities of  cytosine  or  uracil,  it  is  advisable  to  remove  the  excess  of  bro- 
mine by  passing  a  stream  of  air  through  the  solution.  Now  add  an 
excess  of  an  aqueous  solution  of  barium  hydroxide  and  note  the  ap- 
pearance of  a  purple  color. 

Very  dilute  solutions  do  not  give  the  test.  Under  these  conditions 
the  solution  should  be  evaporated  to  dryness,  the  residue  dissolved  in 
a  little  bromine  water  and  the  excess  of  bromine  removed.  Then  upon 
adding  an  excess  of  barium  hydroxide  a  decided  bluish-pink  or  laven- 
der color  will  appear  in  the  presence  of  as  small  an  amount  as  o.ooi 
gram  of  uracil. 

In  testing  solutions  for  cytosine,  it  is  preferable  to  warm  or  boil  the 
solution  with  bromine  water,  and  after  cooling  the  solution  to  apply 
the  test  as  suggested  above,  being  careful  to  have  a  slight  excess  of 
bromine  present  before  adding  barium  hydroxide. 

The  phosphoproteins  are  called  nucleoalhumins  in  many  classifica- 
tions and  are  grouped  among  the  simple  proteins.  They  are  considered 
to  be  "compounds  of  the  protein  molecule  and  some,  as  yet  undefined, 
phosphorus-containing  substances  other  than  a  nucleic  acid  or  lecithin." 
The  percentage  of  phosphorus  in  phosphoproteins  is  very  similar  to 
that  in  nucleoproteins  but  they  differ  from  this  latter  class  of  proteins 
in  that  they  do  not  yield  any  purine  bases  upon  hydrolytic  cleavage. 
Two  of  the  common  phosphoproteins  are  the  caseinogen  of  milk  and 
the  ovovitellin  of  the  egg-yolk. 

The  hemoglobins  (chromoproteins)  are  compounds  of  the  protein 
molecule  with  haematin  or  some  similar  substance.  The  principal 
member  of  the  group  is  the  haemoglobin  of  the  blood.  Upon  hydro- 
lytic cleavage  this  haemoglobin  yields  a  protein  termed  globin  and  a 
coloring  matter  termed  hamo  chroma  gen.  The  latter  substance  con- 
tains iron  and  upon  coming  in  contact  with  oxygen  is  oxidized  to  form 
hcEmatin.  Hcemocyanin,  another  member  of  the  class  of  haemoglobins, 
occurs  in  the  blood  of  certain  invertebrates,  notably  cephalopods, 
gasteropods,  and  Crustacea.  Hasmocyanin  generally  contains  either 
copper,  manganese,  or  zinc  in  place  of  the  iron  of  the  haemoglobin 
molecule. 

The  lecithoproteins  include  such  substances  as  lecithans  and  phos- 

^  Avoid  the  addition  of  a  large  excess  of  bromine  inasmuch  as  this  will  interfere  ^\•ith 
the  test. 


I06  PHYSIOLOGICAL    CHEMIS  fRY. 

phatides  which  consist  of  a  protein  molecule  joined  to  lecithin.  Thev 
have  been  comparatively  little  studied  until  recently,  and  in  much  of 
the  older  research  they  were  undoubtedly  considered  as  lecithins. 

For  experiments  on  conjugated  proteins  see  pages  54,  iqi.  iqy,  198, 
218,  and  224. 

DERIVED  PROTEINS. 

These  substances  are  deri\atives  which  are  formed  through  hydro- 
lytic  changes  of  the  original  protein  molecule.  They  may  be  divided 
into  two  groups,  the  primary  protein  derivatixes  and  the  secondary 
protein  derivatives.  The  term  secondary  derivatives  is  made  use  of 
in  this  connection  since  the  formation  of  the  primary  derivatives  gener- 
ally precedes  the  formation  of  these  secondary  derivatives.  These 
derived  proteins  are  obtained  from  native  simple  proteins  by  hydrol-' 
yses  of  various  kinds,  e.  g.,  through  the  action  of  acids,  alkalis,  heat,  or 
enzymes.  The  particular  class  of  derived  protein  desired  regulates 
the  method  of  treatment  to  which  the  native  protein  is  subjected. 

Primary  Protein  Derivatives. 

The  primary  protein  derivatives  are  "apparently  formed  through 
hydrolytic  changes  which  involve  only  slight  alterations  of  the  protein 
molecule."  This  class  includes  proteans,  metaprotcins,  and  coagulated 
proteins. 

PROTEANS. 

i-*roteans  are  those  insolul)le  protein  substances  whicli  arc  produced 
from  proteins  originally  soluble  through  the  incipient  action  of  water, 
enzymes,  or  very  dilute  acids.  It  is  well  known  that  globulins  become 
insoluble  upon  repeated  reprecipitation  and  it  may  ])ossibly  be  found 
that  the  greater  number  of  the  jjrotcans  are  transformed  globulins. 
Osborne,  howe\'er,  believes  that  nearly  all  ])roteins  may  gi\e  rise  to 
proteans.  This  in\'estigator  wh(j  has  so  \'ery  llioroughl\-  investigated 
many  of  the  vegetable  proteins  claims  thai  the  hydrogen  ion  is  the 
active  agent  in  the  transformation.  The  iJiolein  ])roduced  from  the 
transformati(;n  of  cdcslin  is  called  edeslan,  thai  produced  from  myosin 
is  called  myosan,  etc.  The  name  protean  was  lirst  given  to  this  class 
of  proteins  by  Osborne  in  1900  in  connection  with  his  studies  of  edestin. 

i'',xi'i,i<iMi.\  IS  ON  pR  or  lows. 

Preparation  and  Study  of  Edestan.  Prepare  edestin  according 
to  the  directions  gi\en  on  jjage  101.      Bring  the  edestin  into  solution  in 


PROTEINS.  107 

0.2  per  cent  hydrochloric  acid  and  permit  the  acid  sokition  to  stand  for 
about  one-half  hour/  Neutralize,  with  a  0.5  per  cent  solution  of 
sodium  carbonate,  filter  off  the  precipitate  of  edestan  and  make  the 
following  tests: 

I.  Solubility. — Try  the  solubility  in  the  ordinary  solvents  (see  page 
22).  Note  the  altered  solubility  of  the  edestan  as  compared  with  that 
of  edestin  (see  page  loi). 

2.  Millon's  Reaction. 

3.  Coagulation  Test. — Place  a  small  amount  of  the  protean  in 
a  test-tube,  add  a  little  water  and  boil.  Now  add  dilute  hydrochloric 
acid  and  note  that  the  protein  no  longer  dissolves.  It  has  been 
coagulated. 

4.  Tests  on  Edestan  Solution. — Dissolve  the  remainder  of  the 
edestan  precipitate  in  0.2  per  cent  hydrochloric  acid  and  make  the 
following  tests : 

{a)  Biuret  Test. 

(b)  Infi'iience  of  Protein  Precipitants. — Try  a  few  protein  precipi- 
tants  such  as  picric  acid  and  mercuric  chloride. 

METAPROTEINS. 

The  metaproteins  are  formed  from  the  native  simple  proteins 
through  an  action  similar  to  that  by  which  proteans  are  formed.  In 
the  case  of  the  metaproteins,  however,  the  changes  in  the  original  pro- 
tein molecule  are  more  profound.  These  derived  proteins  are  charac- 
terized by  being  soluble  in  very  weak  acids  and  alkalis,  but  insoluble 
in  neutral  fluids.  The  metaproteins  have  generally  been  termed 
albuminates,  but  inasmuch  as  the  termination  ate  signifies  a  salt  it  has 
always  been  somewhat  of  a  misnomer. 

Two  of  the  principal  metaproteins  are  the  acid  metaprotein  or  so- 
called  acid  albuminate  and  the  alkali  metaprotein  or  so-called  alkali 
albuminate.  They  differ  from  the  native  simple  proteins  principally  in 
being  insoluble  in  sodium  chloride  solution  and  in  not  being  coagulated 
except  when  suspended  in  neutral  fluids.  Both  forms  of  metaprotein 
are  precipitated  upon  the  approximate  neutralization  of  their  solutions. 
They  are  precipitated  by  saturating  their  solutions  with  ammonium 
sulphate,  and  by  sodium  chloride,  also,  provided  they  are  dissolved 
in  an  acid  solution.  x\cid  metaprotein  contains  a  higher  percentage 
of  nitrogen  and  sulphur  than  the  alkali  metaprotein  from  the  same 
source,  since  some  of  the  nitrogen  and  sulphur  of  the  original  protein 
is  liberated  in  the  formation  of  the  latter.     Because  of  this  fact,  it  is 

'  The  edestan  solution  preserved  from  experiment  (5),  page  102,  may  be  used. 


Io8  PHYSIOLOGICAL   CHEMISTRY. 

impossible  to  transform  an  alkali  metaprotein  into  an  acid  metapro- 
tein,  while  it  is  possible  to  reverse  the  process  and  transform  the  acid 
metaprotein  into  the  alkali  modification. 

Experiments  on  Metaproteins. 
acid  metaprotein  (acid  albuminate). 

Preparation  and  Study. — Take  25  grams  of  hashed  lean  beet 
washed  free  from  the  major  portion  of  blood  and  inorganic  matter, 
and  place  it  in  a  medium-sized  beaker  with  100  c.c.  of  0.2  per  cent 
HCl.  Place  it  on  a  boiling  water-bath  for  one-half  hour,  filter,  cool, 
and  divide  the  filtrate  into  two  parts.  Neutralize  the  first  part  with 
dilute  KOH  solution,  filter  ofi'  the  precipitate  of  acid  metaprotein  and 
make  the  following  tests: 

(i)  Solubility. — Solubility  in  the  ordinary  solvents  (see  page  22). 

(2)  Millon's  Reaction. 

(3)  Coagulation  Test. — Suspend  a  little  of  the  metaprotein  in 
water  (neutral  solution)  and  heat  to  boiling  for  a  few  moments.  Now 
add  1-2  drops  of  KOH  solution  to  the  water  and  see  if  the  metapro- 
tein is  still  soluble  in  dilute  alkali.     What  is  the  result  and  why? 

(4)  Test  for  Loosely  Combined  Sulphur  (see  page  100). 

Subject  the  second  part  of  the  original  solution  to  the  following  tests: 
(i)  Coagulatian  Test. — Heat  some  of  the  solution  to  boiling  in  a 
test-tube.    Does  it  coagulate  ? 

(2)  Biuret  Test. 

(3)  Influence  of  Protein  Frccipitants. — Try  a  few  protein  precipi- 
tants  such  as  picric  acid  and  mercuric  chloride.  How  do  the  results 
obtained  compare  with  those  from  the  experiments  on  egg  albumin  ? 
(See  page  95.) 

ALKALI  METAPROTEIN  (ALKALI  ALBUMINATE). 

Preparation  and  Study. — Carefully  separate  the  white  from 
the  yolk  of  a  hen's  egg  and  place  the  former  in  an  evaporating  dish. 
Add  concentrated  potassium  hydroxide  solution,  drop  by  drop,  stirring 
continuously.  The  mass  gradually  thickens  and  finally  assumes 
the  consistency  of  jelly.  This  is  solid  alkali  metaprotein  or  "Licber- 
kijhn's  jelly."  Do  not  add  an  excess  of  potassium  hydroxide  or  the 
jelly  will  dissolve.  Cut  it  into  small  jjieces,  place  a  cloth  or  wire  gau/e 
(jver  the  dish,  and  by  means  of  running  water  wash  the  pieces  free  from 
adherent  alkali.  Now  add  a  small  amount  of  water,  which  forms  a 
xveak  alkaline  solution  with  the  alkali  within  the  pieces,  and  dissolve 


PROTEINS.  109 

the  jelly  by  gentle  heat.  Cool  the  solution  and  divide  it  into  two  parts. 
Proceed  as  follows  with  the  first  part:  Neutralize  with  dilute  hydro- 
chloric acid,  noting  the  odor  of  the  liberated  hydrogen  sulphide  as  the 
alkali  metaprotein  precipitates.  Filter  off  the  precipitate  and  test  as 
for  acid  metaprotein,  page  108,  noting  particularly  the  sulphur  test. 
How  does  this  test  compare  with  that  given  by  the  acid  metaprotein  ? 
Make  tests  on  the  second  part  of  the  solution  the  same  as  for  acid 
metaprotein,  page  108. 

Coagulated  Proteins. 

These  derived  proteins  are  produced  from  unaltered  protein 
materials  by  heat,  by  long  standing  under  alcohol,  or  by  the  con- 
tinuous movement  of  their  solutions  such  as  that  produced  by  rapid 
stirring  or  shaking.  In  particular  instances,  such  as  the  formation  of 
fibrin  from  fibrinogen  (see  page  187),  the  coagulation  may  be  pro- 
duced by  enzyme  action.  Ordinary  soluble  proteins  after  having 
been  transformed  into  the  coagulated  modification  are  no  longer  soluble 
in  the  ordinary  solvents.  Upon  being  heated  in  the  presence  of  strong 
acids  or  alkalis,  coagulated  proteins  are  converted  into  metaproteins. 

Many  proteins  coagulate  at  an  approximately  fixed  temperature 
under  definite  conditions  (see  pp.  98  and  231).  This  characteristic 
may  be  applied  to  separate  different  coagulable  proteins  from  the  same 
solution  by  fractional  coagulation.  The  coagulation  temperature 
frequently  may  serve  in  a  measure  to  identify  proteins  in  a  manner 
similar  to  the  melting-point  or  boiling-point  of  many  other  organic 
substances.  The  separation  of  proteins  by  fractional  coagulation 
is  thus  analogous  to  the  separation  of  volatile  substances  by  means 
of  fractional  distillation.  This  method  of  separating  proteins  is  not 
a  satisfactory  one,  however,  inasmuch  as  proteins  in  solution  have 
different  effects  upon  one  another  and  also  because  of  the  fact  that  the 
nature  of  the  solvent  causes  a  variation  in  the  temperature  at  which 
a  given  protein  coagulates.  The  nature  of  the  process  involved  in 
the  coagulation  of  proteins  by  heat  is  not  well  understood,  but  it  is 
probable  that  in  addition  to  the  altered  arrangement  of  the  component 
atoms  in  the  molecule,  there  is  a  mild  hydrolysis  which  is  accom- 
panied by  the  liberation  of  minute  amounts  of  hydrogen,  nitrogen,  and 
sulphur.  The  presence  of  a  neutral  salt  or  a  trace  of  a  mineral  acid 
may  facilitate  the  coagulation  of  a  protein  solution  (see  page  98). 
whereas  any  appreciable  amount  of  acid  or  alkali  will  retard  or  en- 
tirely prevent  such  coagulation. 


no  PHYSIOLOGICAL    CHEMISTRY. 

Experiments  ox  Coagulated  Proteix. 

Ordinary  coagulated  egg-white  may  l)e  used  in  the  following 
tests: 

1.  Solubility. — Try  the  solubih'ty  of  small  pieces  of  the  coagu- 
lated protein  in  each  of  the  ordinary  sohents  (see  page  22). 

2.  Millon's  Reaction, 

3.  Xanthoproteic  Reaction. — Partly  dissolve  a  medium-sized 
piece  of  the  protein  in  concentrated  nitric  acid.  Cool  the  solution 
and  add  an  excess  of  ammonium  hydroxide.  Both  the  protein  solution 
and  the  undissolved  protein  will  be  colored  orange. 

4.  Biuret  Test. — Partly  dissolve  a  medium-sized  piece  of  the 
protein  in  concentrated  potassium  hydroxide  solution.  If  the  proper 
dilution  of  cupric  sulphate  solution  is  now  added  the  white  coagu- 
lated protein,  as  well  as  the  protein  solution,  will  assume  the  char- 
acteristic purplish-xiolet  color. 

5.  Hopkins-Cole  Reaction.  —Conduct  this  test  according  to  the 
modification  gi\en  on  page  99. 

Secondary  Protein  Derivatives. 

These  derivatives  result  from  a  more  profound  cleavage  of  the 
protein  molecule  than  that  which  occurs  in  the  formation  of  the 
primary  derivati\'es.    The  class  includes  proteoses,  peptones,  and  peptides. 

PROTEOSES  AND  PEPTONES. 

Proteoses  are  intermediate  products  in  the  digestion  of  j)roteins 
by  proteolytic  enzymes,  as  well  as  in  the  decomposition  of  ])roteins 
i)y  hydrolysis  and  the  putrefaction  of  proteins  through  the  action 
of  bacteria.  Proteoses  are  called  also  albumoses  by  some  writers, 
but  it  seems  more  logical  to  reserve  the  term  albumose  for  the  proteose 
of  albumin. 

Pe|)tones  are  formed  after  the  ])roteoses  and  it  has  been  customary 
to  consider  them  as  the  last  jjroduct  oi  the  processes  before  mentioned 
which  still  possess  true  ])rotein  characteristics.  In  other  words  it  has 
been  considered  that  the  protein  nature  of  the  end-products  of  the  cleav- 
age of  the  |>rotein  molecule  ceased  with  the  peptones,  and  that  the  sim- 
pler bodies  formed  from  peptones  were  substances  of  a  different  nature 
I'see  page  65).  However,  as  the  end-jjrodiK  is  ha\e  been  more  carefully 
studied,  it  has  been  found  to  be  no  easy  matter  to  designate  the  exact 
character  of  a   j)e|)tone  or  to  indicate  the  exuct  ])(jint  at  which  the 


PBOTEINS.  Ill 

peptone  characteristic  ends  and  the  peptide  characteristic  begins.  The 
situation  regarding  the  proteoses,  peptones  and  peptides,  is  at  present 
a  most  unsatisfactory  one  because  of  the  unsettled  state  of  our  knowl- 
edge regarding  them.  The  exact  differences  between  certain  members 
of  the  peptone  and  peptide  groups  remain  to  be  more  accurately 
established.  It  has  been  quite  well  established  that  the  peptones  are 
peptides  or  mixtures  of  peptides,  but  the  term  peptide  is  used  at 
present  to  designate  only  those  possessing  a  definite  structure. 

There  are  several  proteoses  (protoproteose;  heteroproteose  and 
deuteroproteose),  and  at  least  two  peptones  (amphopeptone  and 
antipeptone),  which  result  from  proteolysis.  The  differentiation 
of  the  various  proteoses  and  peptones  at  present  in  use  is  rather  unsatis- 
factory. These  compounds  are  classified  according  to  their  varying 
solubilities,  especially  in  ammonium  sulphate  solutions  of  different 
strengths.  The  exact  differences  in  composition  between  the  various 
members  of  the  group  remain  to  be  more  accurately  established. 
Because  of  the  difficulty  attending  the  separation  of  these  bodies, 
pure  proteose  and  peptone  are  not  easy  to  procure.  The  so-called 
peptones  sold  commercially  contain  a  large  amount  of  proteose.  As 
a  class  the  proteoses  and  peptones  are  very  soluble,  diffusible  bodies 
which  are  non-coagulable  by  heat.  Peptones  differ  from  proteoses  in 
being  more  diffusible,  non-precipitable  by  (NHJ^SO^,  and  by  their 
failure  to  give  any  reaction  with  potassium  ferrocyanide  and  acetic  acid, 
potassio-merciiric  iodide  and  HCl,  picric  acid,  and  trichloracetic  acid. 
The  so-called  primary  proteoses  are  precipitated  by  HNO3  and 
are  the  only  members  of  the  proteose-peptone  group  which  are  so 
precipitated. 

Some  of  the  more  general  characteristics  of  the  proteose-peptone 
group  may  be  noted  by  making  the  following  simple  tests  on  a  proteose- 
peptone  powder: 

(i)  Solubility. — Solubility  in  the  ordinary  solvents  (see  page  22). 

(2)  Mil  Ion  ^s  Reaction. 

Dissolve  a  little  of  the  powder  in  water  and  test  the  solution  as 
follows: 

(i)  Precipitation  by  Picric  Acid. — To  5  c.c.  of  proteose-peptone 
solution  in  a  test-tube  add  picric  acid  until  a  permanent  precipitate 
forms.    The  precipitate  disappears  on  heating  and  returns  on  cooling. 

(2)  Precipitation  by  a  Mineral  Acid. — Try  the  precipitation  by 
nitric    acid. 

(3)  Coagulation  Test. — Heat  a  little  proteose-peptone  solution 
to  boiling.     Does  it  coagulate  like  the  other  simple  proteins  studied? 


112  PHYSIOLOGICAL    CHEMISTRY. 

SEPARATION  OF  PROTEOSEvS  AND  PEPTONES.' 

Place  50  c.c.  of  protcose-peptonc  solution  in  an  exaporating  dish 
or  casserole,  and  half-saturate  it  with  ammonium  sulphate  solution, 
which  may  be  accomplished  by  adding  an  equal  ^■olume  of  saturated 
ammonium  sulphate  solution.  At  this  point  note  the  appearance 
of  a  precipitate  of  the  primary  proteoses  (protoproteose  and  hetero- 
proteose).  Now  heat  the  half-saturated  solution  and  its  suspended 
precipitate  to  boiling  and  saturate  the  solution  with  solid  ammonium 
sulphate.  At  full  saturation  the  secondary  proteoses  (deuteroproteoscs) 
are  precipitated.    The  peptones  remain  in  solution. 

Proceed  as  follows  with  the  precipitate  of  proteoses:  Collect  the 
sticky  precipitate  on  a  rubber-tipped  stirring  rod  or  remove  it  by 
means  of  a  watch  glass  to  a  small  evaporating  dish  and  dissolve  it  in  a 
little  water.  To  remove  the  ammonium  sulphate,  which  adhered  to 
the  precipitate  and  is  now  in  solution,  add  barium  carbonate,  boil, 
and  filter  ofT  the  precipitate  of  barium  sulphate.  Concentrate  the 
proteose  solution  to  a  small  volume^  and  make  the  following  tests: 

(i)  Biuret  Test. 

(2)  Precipitation  by  Nitric  Acid. — What  would  a  precipitate  at 
this  point  indicate? 

(3)  Precipitation  by  Trichloracetic  Acid. — This  precipitate  dis- 
solves on  heating  and  returns  on  cooling. 

(4)  Precipitation  by  Picric  Acid. — This  precipitate  also  disap- 
pears on  heating  and  returns  on  cooling. 

(5)  Precipitation  by  Potassio-mercuric  Iodide  and  Hydrochloric 
Acid. 

(6)  Coagulation  Test. — Boil  a  little  in  a  test-tube.  Does  it 
coagulate  ? 

(7)  Acetic  Acid  mid  Potassium  Ferrocyanide  Test. 

The  solution  containing  the  jjeptones  should  be  cooled  and  fil- 
tered, and  the  ammonium  sulphate  in  solution  removed  by  boiling 
with  barium  carbonate  as  described  above.  After  filtering  off  the 
inirium  sulphate  precipitate,  concentrate  the  peptone  filtrate  to  a 
small  volume  and  repeat  the  test  as  given  under  the  proteose  solu- 
tion, ab(ne.  In  the  biuret  test  the  solution  should  be  made  very 
strongly  alkaline  with  solid  potassium  hydroxide. 

'  'I'hc  S(j|);iration  of  jirutcoscs  riml  |i(|i|iiiics  \iy  iniriins  of  fiartioiial  |)rc<;i|)iLali<in  wilh 
amn)')niuni  sulpliato  doc-s  not  jjosscss  Uic  si^^nit'ii  aii(  c  it  ont  c  fjossisscd  inasmuch  as  tlie 
boundary  between  tiicse  substances  and  peptides  is  not  well  I'efmed  (see  f).  110). 

'  If  the  proteoses  are  <lesired  in  povvder  form,  this  com cntraletl  piolt-ose  solution  may 
now  be  [jrei  ijjitalefl  by  aUolioi,  anfl  this  jirecipitatc,  :ifl<T  Ixin^^  waslicd  with  absolute 
alcohol  anrl  with  ether,  may  be  rlried  and  ]K)W<lcr(!fl. 


PROTEINS. 
PEPTIDES. 


113 


The  peptides  are  "definitely  characterized  combinations  of  two 
or  more  amino  acids,  the  carboxyl  (COOH)  group  of  one  being 
united  with  the  amino  (NHJ  group  of  the  other  with  the  elimination 
of  a  molecule  of  water."  These  peptides  are  more  fully  discussed  on 
pages  65  and  iii. 

REVIEW  OF   PROTEINS. 

In  order  to  facilitate  the  student's  review  of  the  proteins,  the 
preparation  of  a  chart  similar  to  the  model  given  is  recommended. 
The  signs  +  and  —  may  be  conveniently  used  to  indicate  positive 
and  negative  reactions. 


MODEL  CHART  FOR  REVIEW  PURPOSES. 


Protein. 

Solubility. 

e 

u 
"o 

0 

c 

0 

Salting- 
Precipitation  Tests.                 out 
Tests. 

.  (U    • 

1 
1 

10%  NaCl. 
0.2%  HCl. 

0    1  .^-   '  tri 
0 

3' 
'0    . 

0 

0 
0 

< 

•u    . 

■a:2 

cS  0 

><< 

0  0 

^J 

a, 

Potassio-mercuric 
Iodide -1- HCl. 

Picric  Acid. 

Trichloracetic 
Acid. 

(NH4)2SO.i. 

^ 

S 

_3 
3' 

5 

(3    . 

■  § 
5i 

Albumin. 

— 



— 

— 

Globulin. 





Protean. 

Acid  metaprotein. 



Alkali  metaprotein. 

' 

Proteose. 
Peptone. 

: 

;  ' 

Coagulated  protein.    |        j       1 

! 

, 

"Unknown"  Mixtures  and  Solutions  of  Proteins. 

At  this  point  the  student's  knowledge  of  the  characteristics  of 
the  various  proteins  studied  will  be  tested  by  requiring  him  to  ex- 
amine several  "unknown"  protein  mixtures  or  solutions  and  make 
full  report  upon  the  same.  The  scheme  given  on  page  114  may  be 
used  in  this  examination. 


114 


PHYSIOLOGICAL    CHEMISTRY. 


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CHAPTER  VI . 
GASTRIC  DIGESTION. 

Gastric  digestion  takes  place  in  the  stomach  and  is  promoted  by 
the  gastric  juice,  which  is  secreted  by  the  glands  of  the  stomach 
mucosa.  These  glands  are  of  two  kinds,  fundus  glands  and  pyloric 
glands  which  are  situated,  as  their  names  imply,  in  the  regions  of  the 
fundus  and  pylorus.  The  principal  foods  acted  upon  in  gastric 
digestion  are  the  proteins  which  are  so  changed  by  its  processes  as 
to  become  better  prepared  for  further  digestion  in  the  intestine  and 
for  their  final  absorption. 

From  reliable  experiments  made  upon  lower  animals  it  is  evident 
that  the  gastric  juice  is  secreted  as  the  result  of  stimuli  of  two  forms, 
/.  e.,  psychical  stimuli  and  chemical  stimuli.  The  psychical  form  of 
stimuli  may  be  produced  by  the  sight,  thought,  or  taste  of  food,  and 
the  chemical  stimuli  may  be  produced  by  certain  substances,  such  as 
water,  the  extractives  of  meat,  etc.,  when  coming  in  contact  with  the 
stomach  mucosa.  The  volume  of  gastric  juice  secreted  during  any 
given  period  of  digestion,  varies  with  the  quantity  and  kind  of  the 
food.  These  conclusions  were  deduced  principally  from  a  series  of 
so-called  delusive  feeding  experiments.  A  dog  was  prepared  with  two 
oesophageal  openings  and  a  gastric  fistula.  When  thus  prepared  and 
fed  foods  of  various  kinds  such  as  meat  and  bread,  the  material  instead 
of  passing  to  the  stomach,  would  invariably  find  its  way  out  of  the 
animal 's  body  at  the  upper  oesophageal  opening.  Through  the  medium 
of  the  gastric  fistula  the  course  of  the  secretion  of  gastric  juice  could 
be  carefully  followed.  It  was  found  that  when  the  dog  ate  meat,  for 
example,  there  was  a  large  secretion  of  gastric  juice  notwithstanding 
no  portion  of  the  food  eaten  had  reached  the  stomach.  Further 
experiments  made  through  the  medium  of  a  cul-de-sac  formed  from 
the  stomach  wall  have  given  us  many  valuable  conclusions,  among 
others  those  regarding  the  influence  of  the  chemical  stimuli.  The 
method  followed  was  to  feed  the  animal  certain  substances  and  note 
the  secretion  of  gastric  juice  in  the  miniature  stomach  while  the  real 
process  of  digestion  was  taking  place  in  the  stomach  proper. 

Normal  gastric  juice  is  a  thin,  light  colored  fluid  which  is  acid 

115 


Il6  PHYSIOLOGICAL    CHEAUSTRY. 

in  reaction  and  has  a  specific  gravity  varying  between  i.ooi  and  i.oio. 
It  contains  only  2-3  per  cent  of  solid  matter  which  is  made  up  prin- 
cipally of  hydrochloric  acid,  sodium  chloride,  potassium  chloride, 
earthy  phosphates,  mucin  and  the  enzymes  pepsin,  gastric  rennin,  and 
gastric  lipase;  the  hydrochloric  acid  and  the  enzymes  are  of  the  greatest 
importance.  The  acidity  of  the  gastric  juice  is  due  to  free  hydrochloric 
acid  which  is  secreted  by  the  parietal  cells  of  the  fundus  as  well  as  by 
the  chief  cells  of  both  the  fundus  and  pyloric  glands,  and,  in  man,  is 
generally  present  to  the  extent  of  0.2-0.3  per  cent.  When  the  amount 
of  hydrochloric  acid  varies  to  any  considerable  degree  from  these 
values  a  condition  of  hypoacidity  or  hyperacidity  is  established. 
Hydrochloric  acid  has  the  power  of  combining  with  protein  substances 
taken  in  the  food,  thus  forming  so-called  combined  hydrochloric  acid. 
This  combined  acid  is  a  less  potent  germicide  than  free  hydrochloric 
acid  and  has  less  power  to  destroy  the  amylolytic  enzyme  salivary 
amylase  (ptyalin)  of  the  saliva.  This  last  fact  explains  to  a  degree  the 
possibility  of  the  continuance  of  salivary  digestion  in  the  stomach. 
The  hydrochloric  acid  of  the  gastric  juice  forms  a  medium  in  which 
the  pepsin  can  most  satisfactorily  digest  the  protein  food,  and  at  the 
same  time  it  acts  as  an  antiseptic  or  germicide  which  prevents  putre- 
factive processes  in  the  stomach.  It  also  possesses  the  power  of  in- 
verting cane  sugar,  this  property  being  due  to  the  hydrogen  ion.  When 
the  hydrochloric  acid  of  the  gastric  juice  is  diminished  in  (juantity 
(hypoacidity)  or  absent,  as  it  may  be  in  many  cases  of  functional  or 
organic  disease,  there  is  no  check  to  the  growth  of  micro-organisms 
in  the  stomach.  There  are,  however,  certain  of  the  more  resistant 
spores  which  even  the  normal  acidity  of  the  gastric  juice  will  not  de- 
stroy. A  condition  of  hypoacidity  may  also  give  rise  to  fermentation 
with  the  formation  of  comparatively  large  amounts  of  such  substances 
as  lactic  acid  and  butyric  acid. 

The  (jucstion  of  the  origin  of  the  hydrochloric  acid  of  the  gastric 
juice  is  a  problem  to  whose  solution  many  investigators  have  given 
much  attention.  Many  theories  have  been  proposed,  among  them 
being  iiunge's  mass  action  theory,  Koppe's  electrolytic  dissociation 
theory,  and  the  more  recent  theory  based  upon  the  interaction  of  sodium 
chloride  and  lactic  acid.  We  cannot  go  into  a  discussion  of  these  various 
theories.  Each  of  them  has  met  with  objection  and  we  have,  as  yet, 
no  generally  accepted  theory  as  to  the  origin  of  the  hydrochloric  acid 
of  the  gastric  juice.  That  this  hydrochloric  acid  originates  from  the 
chlorides  of  the  blood  is  apparently  a  well  established  fact,  but  farther 
than  this  no  positive  statement  can  be  made. 


GASTRIC    DIGESTION.  II7 

The  most  important  of  the  enzymes  of  the  gastric  juice  is  the 
proteolytic  enzyme  pepsin.  The  pepsin  does  not  originate  as  such 
in  the  gastric  cells  but  is  formed  from  its  precursor,  the  zymogen  or 
mother-substance  pepsinogen,  which  is  produced  by  the  parietal  cells 
of  the  fundus  as  well  as  by  the  chief  cells  of  the  fundus  and  pyloric 
glands.  Upon  coming  in  contact  with  the  hydrochloric  acid  of  the 
secretion  this  pepsinogen  is  immediately  transformed  into  pepsin. 
Pepsin  is  not  active  in  alkaline  or  neutral  solutions  but  requires  at 
least  a  faint  acidity  before  it  can  exert  its  power  to  dissolve  and  digest 
proteins.  The  percentage  of  hydrochloric  acid  facilitating  the  most 
rapid  peptic  action  varies  with  the  character  of  the  protein  acted  upon, 
e.  g.,  0.08  per  cent  to  o.i  per  cent  for  the  digestion  of  fibrin  and  0.25 
per  cent  for  the  digestion  of  coagulated  egg-white.  While  hydrochloric 
acid  is  the  acid  usually  employed  to  promote  artificial  peptic  pro- 
teolysis, other  acids,  organic  and  inorganic,  will  serve  the  same 
purpose.  x\cidity  of  the  liquid  is  necessary  to  promote  the  activity 
of  the  pepsin,  but  the  acidity  need  not  necessarily  be  confined  to 
hydrochloric   acid. 

In  common  with  many  other  enzymes  pepsin  acts  best  at  about 
38°-4o°  C.  and  its  digestive  power  decreases  as  the  temperature  is 
lowered,  the  enzyme  being  only  slightly  active  at  0°  C.  Its  power  is 
only  temporarily  inhibited  by  the  application  of  such  low  temperatures, 
however,  and  the  enzyme  regains  its  full  proteolytic  power  upon  raising 
the  temperature  to  40°  C.  As  the  temperature  of  a  digestive  mixture 
is  raised  above  40°  C.  the  pepsin  gradually  loses  its  activity  until  at 
about  8o°-ioo°  C.  its  proteolytic  power  is  permanently  destroyed. 

Our  ideas  regarding  the  nature  of  the  products  formed  in  the 
course  of  peptic  proteolysis  have  undergone  considerable  revision 
in  recent  years.  The  former  view  that  these  products  included  only 
acid  albuminate  (acid  metaprotein),  proteoses  and  peptones  is  no 
longer  tenable.  From  the  investigations  of  numerous  observers  we 
have  learned  that  artificial  gastric  digestion  if  permitted  to  proceed  for 
a  sufficiently  long  period  will  yield,  in  addition  to  proteoses  and  pep- 
tones, a  long  list  of  protein  cleavage  products  which  are  crystalline 
in  character,  including  leucine,  tyrosine,  alanine,  phenylalanine,  aspartic 
acid,  glutamic  acid,  proline,  leucinimide,  valine,  and  lysine.  A  similar 
group  of  substances  may  result  from  the  action  of  the  enzyme  trypsin 
(see  p.  138).  The  relative  amounts  of  proteoses,  peptones,  and  crys- 
talline substances  formed  depends  to  a  great  extent  upon  the  character 
of  the  protein  undergoing  digestion,  e.  g.,  a.  greater  proportion  of  pro- 
teoses results  from  the  digestion  of  fibrin  than  from  the  digestion  of 


Il8  PHYSIOLOGICAL    CHEMISTRY. 

coagulated  egg-white.  We  must  not  be  led  into  the  error  of  thinking 
that  the  large  number  of  protein  cleavage  products  just  mentioned 
are  formed  in  the  course  of  normal  gastric  digestion  zci/Jihi  the  animal 
organism.  They  are  formed  only  after  comparatively  long-continued 
hydrolysis.  In  pancreatic  digestion,  however,  there  are  formed  even 
under  normal  conditions,  the  large  number  of  cleavage  products 
to  which  reference  has  been  made.  Peptic  proteolysis,  therefore, 
within  the  animal  organism  differs  from  tryptic  proteolysis  (see  page 
138)  in  that  the  former  yields  larger  amounts  of  proteoses,  smaller 
amounts  of  peptones  and  no  considerable  (|uantity  of  crystalline 
bodies  as  end-products  in  the  brief  period  during  which  proteins  are 
ordinarily  subjected  to  gastric  digestion.  Prolonged  hydrolysis  with 
gastric  juice  does,  however,  yield  considerable  quantities  of  the  non- 
protein end-products. 

Gastric  rennin,  the  second  enzyme  of  the  gastric  juice,  is  what  is 
known  as  a  milk  curdling  or  protein  coagulating  enzyme.  Rennin 
acts  upon  the  caseinogen  of  the  milk,  splitting  it  into  a  proteose-like 
body  and  soluble  casein.  This  soluble  body,  in  the  presence  of  calcium 
salts,  combines  with  calcium,  forming  calcium  casein  or  true  casein 
which  is  insoluble  and  precipitates.  There  is  some  uncertainty  re- 
garding the  reaction  to  litmus  in  which  gastric  rennin  shows  the  great- 
est actixity.  It  is,  however,  said  to  be  active  in  neutral,  alkaline,  or 
acid  solution.  However,  it  probably  possesses  its  greatest  activity  in 
the  presence  of  a  slight  acid  reaction,  as  would  naturally  be  expected. 
It  is  especially  abundant  in  the  gastric  mucosa  of  the  calf,  and  is  used 
to  curdle  the  milk  used  in  cheese-making.  Ciastric  rennin  is  always 
present  normally  in  the  gastric  juice  but  in  certain  pathological  con- 
ditions such  as  atrophy  of  the  mucosa,  chronic  catarrh  of  the  stomach, 
or  in  carcinoma  it  may  be  absent. 

The  theory  that  the  proteolytic  activity  and  the  milk  curdling 
property  of  the  gastric  juice  reside  in  a  single  molecule  is  causing 
much  controversy  at  the  present  time.  'Ihc  theory  was  originally 
adNanced  Ijy  the  Pavvlow  school. 

Gastric  lipase,  the  third  enzyme  ot"  the  gastric  juice,  is  a  fat-splitting 
enzyme.  It  [)ossesses  but  slight  acti\ity  when  the  gastric  juice  is  of 
normal  acidity,  but  evinces  its  action  princi[)ally  at  such  times  as  a 
gastric  juice  of  low  acidity  is  secreted  either  from  jjhysiological  or 
pathological  cause.  The  digestion  of  fat  in  the  stomach  is,  however, 
at  most,  of  but  slight  im|)ortance  as  compared  willi  the  digestion  of 
fat  in  the  intestine  through  the  action  of  the  lipiisc  of  the  pancreatic 
juice  (see  page    140). 


GASTRIC    DIGESTION.  II9 

PREPARATION  OF  AN  ARTIFICIAL  GASTRIC  JUICE. 

Dissect  the  mucous  membrane  of  a  pig's  stomach  from  the  mus- 
cular portion  and  discard  the  latter.  Divide  the  mucous  membrane 
into  two  parts  (4/5  and  1/5).  Cut  up  the  larger  portion,  place  it  in 
a  large-sized  beaker  with  0.4  per  cent  hydrochloric  acid  and  keep 
at  38°-4o°  C.  for  at  least  24  hours.  Filter  off  the  residue,  consisting 
principally  of  nuclein  and  anti-albumid,  and  use  the  filtrate  as  an 
artificial  gastric  juice.  This  filtrate  contains  pepsin,  rennin,  and  the 
products  of  the  digestion  of  the  stomach  tissue,  /.  e..  acid  metaprotein 
(acid  albuminate),  proteoses,  peptones,  etc. 


Preparation  of  a  Glycerol  Extr.a.ct  of  Pig's  Stomach. 

Take  the  one-fifth  portion  of  the  mucous  membrane  of  the  pig's 
stomach  not  used  in  the  preparation  of  the  artificial  gastric  juice, 
cut  it  up  very  finely,  place  it  in  a  small-sized  beaker  and  cover  the 
membrane  with  glycerol.  Stir  frequently  and  allow  to  stand  at  room 
temperature  for  at  least  24  hours.  The  glycerol  will  extract  the  pep- 
sinogen. Separate,  with  a  pipette  or  by  other  means,  the  glycerol 
from  the  pieces  of  mucous  membrane  and  use  the  glycerol  extract  as 
required  in  the  later  experiments. 

Products  of  Gastric  Digestion. 

Into  the  artificial  gastric  juice,  prepared  as  above  described,  place 
the  protein  material  (fibrin,  coagulated-  egg-white,  or  lean  beef) 
provided  for  you  by  the  instructor,  add  0.4  per  cent  hydrochloric 
acid  as  suggested  by  the  instructor  and  keep  the  digestion  mixture  at 
40°  C.  for  2  to  3  days.  Stir  frequently  and  keep /re'?  hydrochloric  acid 
present  in  the  solution  (for  tests  for  free  hydrochloric  acid  see  p.  120). 

The  original  protein  has  been  digested  and  the  solution  now  con- 
tains the  products  of  peptic  proteolysis,  /.  e.,  acid  metaprotein  (acid 
albuminate),  proteoses,  peptones,  etc.  The  insoluble  residue  may 
include  nuclein  and  anti-albumid.  Filter  the  digestive  mixture  and 
after  testing  for  free  hydrochloric  acid  neutralize  the  filtrate  v.-ith 
potassium  hydroxide  solution.  If  any  of  the  acid  metaprotein  (acid 
albuminate)  is  still  untransformed  into  proteoses  it  will  precipitate 
upon  neutralization.  If  any  precipitate  forms  heat  the  mixture  to 
boiling,  and  filter.     If  no  precipitate  forms  proceed  without  filtering. 


I20  PHYSIOLOGICAL    CHEMISTRY. 

We  now  have  a  solution  containing  a  mixture  consisting  princi- 
pally of  proteoses  and  peptones.  Separate  and  identify  the  pro- 
teoses and  peptones  according  to  the  directions  given  on  pages  no 
and  112. 

Tests  for  Free  and  Combined  HCl. 

These  tests  are  made  with  a  class  of  reagents  known  as  indicators, 
so  called  because  they  serve  to  indicate  the  nature  of  the  reaction  of 
a  solution.  These  indicators  are  weak  acids  or  bases  and  are  but 
slightly  dissociable.  The  dissociation,  with  the  formation  of  the 
colored  ion.  forms  the  basis  for  the  color  reaction. 

Examine  each  of  the  following  solutions  by  means  of  the  tests  given 
i>elow  and  report  the  results  in  a  form  similar  to  the  chart  given  on 
page  122:  (i)  0.2  per  cent  free  hydrochloric  acid.  (2)  0.05  per  cent 
free  hydrochloric  acid.  (3)  o.oi  per  cant  free  hydrochloric  acid.  (4) 
0^05  per  cent  combined  hydrochloric  acid.  (5)  i  per  cent  lactic  acid. 
(6)  Equal  volumes  of  0.2  per  cent  free  hydrochloric  acid  and  i  per  cent 
lactic  acid.     (7)  i  per  cent  potassium  hydroxide. 

1.  Dimethyl-amino-azobenzene  (or  Topfer's  Reagent),* 

N(CH3),-C„H,-N  =  N-C„H3. 

Place  1-2  drops  of  the  reagent  in  the  solution  to  Ije  tested.  Free 
mineral  acid  (hydrochloric  acid)  is  indicated  by  the  production  of  a 
I^inkish-red  color.  If  free  acid  is  absent  a  yellow  color  ordinarily 
results. 

2.  Gunzberg's  Reagent.-  Place  1-2  drops  of  the  reagent  in  a 
small  porcelain  evaporating  dish  and  carefully  evajjorale  to  dryness 
over  a  low  flame.  Insert  a  glass  stirring  rod  into  the  mixture  to  be 
tested  and  draw  the  moist  end  of  the  rod  through  the  dried  reagent. 
Warm  again  gently  and  note  the  production  of  a  purplish-red  color 
in  the  presence  <  A  free  hydrochloric  acid. 

3.  Boas'  Reagent.''  Perform  this  lest  in  the  .same  manner  as 
-',  abo\e.  I'rcc  hydrochloric  acid  is  indicated  by  the  production 
oi  a  rose  red  cfjlor  which  becomes  less  pronounced  on  cooling. 

' 'I"o  pre|>are  'I'opfcr's  rcaj^ciit  dissolve  0.5  ^;r;uii  of  cli-iticiliyl-ainino-azDhcn/A-iu;  in 
100  (  .<:.  of  95  j)(.T  cent  alcohol. 

-  (ill^zl)cr^'s  rcagt-nt  is  ))rc'|)arc<]  tiy  dissolving  2  grams  of  pliloioglvn  in  .ind  1  gram 
of  vanillin  in  100  <  .<:.  of  95  jjer  cent  alcohol. 

■^  Boas'  reagent  is  [)re|)arerl  by  dissolving  5  grams  of  rcson  in  and  •;  grams  of  sucrose 
in  100  t.c.  of  9t  per  (ent  alcohol. 


GASTRIC    DIGESTION.  121 

4.  Congo  Red,^ 

NH,  S03Na 


\ 


lN=r:N<'  ><r  >N=N 

\/   \/  \_.  _/  \ /  \/    \. 

SO,Na  NH 


3-^ 


Conduct  this  test  according  to  the  directions  given  under  i  or  2,  page 
120.  A  blue  color  indicates  free  hydrochloric  acid,  a  violet  color 
indicates  an  organic  acid  and  a  brown  color  indicates  combined  hydro- 
chloric acid.  Congo-red  paper,  made  by  immersing  ordinary  filter 
paper  in  the  indicator  and  subsequently  drying,  may  be  used  in  this 
test. 

5.  Tropaeolin  00,^ 

NH(C,H5)-C6H,-N  =  N-CeH,-S03Na. 

Place  2  drops  of  the  solution  to  be  tested  and  i  drop  of  the  indicator 
in  an  evaporating  dish  and  evaporate  to  dryness  over  a  low  flame. 
The  formation  of  a  reddish-violet  color  indicates  free  hydrochloric 
acid. 

This   test   may    also   be   conducted   in   the   same   manner   as    2, 
page  120. 


6.  Phenolphthalein,^ 


C„H.OH 


C— CgH.OH 


CeH,         O 


C 

\ 
O 


Add  the  indicator  directly  to  the  solution,  or  apply  the  test  according 
to  the  directions  given  under  2  on  page  120.  This  indicator  serves 
to  denote  the  total  acidity  since  it  is  acted  upon  by  free  mineral  acids, 
combined  acids,  organic  acids,  and  acid  salts.     A  red  color  indicates  the 

'  This  indicator  is  prepared  by  dissolving  0.5  gram  of  congo  red  in  90  c.c.  of  water 
and  adding  10  c.c.  of  95  per  cent  alcohol. 

'Prepared  by  dissolving  0.05  gram  of  tropaeolin  OO  in  100  c.c.  of  50  per  cent  alcohol. 

^  This  indicator  is  prepared  by  dissolving  i  gram  of  phenolphthalein  in  100  c.c.  of  95 
per  cent  alcohol. 


122 


PHYSIOLOGICAL    CHEMISTRY. 


presence  of  an  alkali  and  the  indicator  is  colorless  in  the  presence  of  a 
neutral  or  acid  reaction.  This  indicator  is  unsatisfactory  in  the  pres- 
ence of  ammonia. 

7.  Sodium  Alizarin  Sulphonate,^ 

CO  (OH), 


CoH, 


an 


\ 


CO 


SO.,Xa 


This  indicator  may  be  used  directly  in  the  solution  to  be  tested,  or 
the  test  may  be  applied  as  2,  page  120.  It  serves  to  indicate  all  acid 
reactions  except  those  due  to  combined  acids.  A  reddish-violet  color 
indicates  an  alkaline  reaction,  while  a  yellow  color  indicates  an  acid 
reaction  due  to  a  free  mineral  acid,  an  organic  acid,  or  an  acid  salt. 
Report  the  results  of  your  tests  tabulated  in  the  form  given  below: 


Name  ot  Indicator. 


Topfers  Keagcni. 
Gunzberg's  Reagent 
Boas'  Reagent. 
Congo  Red. 
Tropaeolin  OO. 
PhenolphthaleJn. 
.Alizarin. 


Solutions  Examined. 

0.2%        0.05%    '   0.01% 
HCl.          HCl.      ;     HCl. 

1        ! 

0.05%         1% 
Combined     Lactic 
HCl.            Acid. 

Equal  Vols. 
0.2%  HCl 

and  1% 
Lactic  Acid. 

1% 
KOH. 

1 

1 

1                1 

1                1                1                1 

i                1                1 

GENERAL  EXPERIMENTS  ON  GASTRIC  DIGESTION. 

I.  Conditions  Essential  for  the  Action  of  Pepsin. — Prepare 
four  test  tubes  as  follows: 

(a)  Five  c.c.  of  pepsin  solution. 
ib)  F'ive  c.c.  of  0.4  per  cent  hydrochloric  acid. 
{c)  Five  c.c.  of  pepsin-hydrochloric  acid  solution. 
(d)  Two  or  three  c.c.  of  jK-psin  solution  and  2    i^  c.c.  of  0.5  per 
cent  sodium  carb(jnate  solution. 


'  Frejwre  this  indiialor  l>y  'lissolving  i  gnini  of  sodium  ;ili/;irni  sul|)lion;iti:  in  joo  c 
fif  water. 


GASTRIC    DIGESTION.  1 23 

Introduce  into  each  tube  a  small  piece  of  iibrin  and  place  them 
on  the  water-bath  at  40°  C.  for  one-half  hour,  carefully  noting  any 
changes  which  occur/  Now  combine  the  contents  of  tubes  (a)  and 
(b)  and  see  if  any  further  change  occurs  after  standing  at  40°  C.  for 
1 5-20  minutes.    Explain  the  results  obtained  from  these  five  experiments. 

2.  Influence  of  Different  Temperatures. — In  each  of  four  test- 
tubes  place  5  c.c.  of  pepsin-hydrochloric  acid  solution.  Immerse 
one  tube  in  cold  water  from  the  faucet,  keep  a  second  tube  at  room 
temperature  and  place  a  third  on  the  water-bath  at  40°  C.  Boil  the 
contents  of  the  fourth  tube  for  a  few  moments,  then  cool  and  also 
keep  it  at  40°  C.  Into  each  tube  introduce  a  small  piece  of  fibrin  and 
note  the  progress  of  digestion.  In  which  tube  does  the  most  rapid 
digestion  occur?     Explain  this. 

3.  The  Most  Favorable  Acidity. — Prepare  three  tubes  as  follows: 

(a)  Five  c.c.   of  0.2   per  cent  pepsin-hydrochloric  acid  solution. 

(b)  Two  or  three  c.c.  of  0.2  per  cent  hydrochloric  acid  -\-  i  c.c. 
of  concentrated  hydrochloric  acid  +  5  c.c.  of  pepsin  solution. 

(c)  One  c.c.  of  0.2  per  cent  pepsin-hydrochloric  acid  solution 
-f  5  c.c.  of  water. 

Introduce  a  small  piece  of  fibrin  into  each  tube,  keep  them  at 
40°  C,  and  note  the  progress  of  digestion.  In  w^hich  degree  of  acidity 
does  the  fibrin  digest  the  most  rapidly  ? 

4.  Differentiation  Between  Pepsin  and  Pepsinogen. — Prepare 
five  tubes  as  follows: 

(a)  Few  drops  of  glycerol  extract  of  pepsinogen  -!-  2-3  c.c.  of 
water. 

(b)  Few  drops  of  glycerol  extract  of  pepsinogen  -f-  5  c.c.  of  0.2 
per  cent  hydrochloric  acid. 

(c)  Few  drops  of  glycerol  extract  of  pepsinogen  -j-  5  c.c.  of  0.5 
per  cent  sodium  carbonate. 

(d)  Two  or  three  c.c.  of  pepsin  solution  -f  2-3  c.c.  of  i  per  cent 
sodium  carbonate. 

(e)  Few  drops  of  glycerol  extract  of  pepsinogen  +  5  c.c.  of  i 
per  cent  sodium  carbonate. 

Add  a  small  piece  of  fibrin  to  the  contents  of  each  tube,  keep 
the  five  tubes  at  40°  C.  for  one-half  hour  and  observe  any  changes 

^  Digestion  of  fibrin  in  a  pepsin-hydrochloric  acid  solution  is  indicated  first  by  a  s^ccelling 
of  the  protein  due  to  the  action  of  the  acid,  and  later  by  a  disintegration  and  dissolving  of 
the  fibrin  due  to  the  action  of  the  pepsin-hydrochloric  acid.  If  uncertain  at  any  time 
whether  digestion  has  taken  place,  the  solution  under  examination  may  be  filtered  and  the 
biuret  test  applied  to  the  filtrate.  A  positive  reaction  will  signify  the  presence  of  acid 
metaprotein  (acid  albuminate),  proteoses  (albumoses),  or  peptones,  the  presence  of  anyone 
of  which  would  indicate  that  digestion  has  taken  place. 


124  PHYSIOLOGICAL    CHEMISTRY. 

which  may  have  occurred.  To  [a)  add  an  equal  volume  of  0.4  per 
cent  hydrochloric  acid,  neutralize  (c),  ((/)  and  [c]  with  hydrochloric 
acid  and  add  an  equal  volume  of  0.4  per  cent  hydrochloric 
acid.  Place  these  tubes  at  40°  C.  again  and  note  any  further  changes 
which  may  occur.  What  contrast  do  we  find  in  the  results  from  the 
last  three  tubes  ?    Why  is  this  so  ? 

5.  Comparative  Digestive  Power  of  Pepsin  with  Different 
Acids. — Prepare  a  scries  of  tubes  each  containing  one  of  the  following' 
acids:  0.5  per  cent  acetic,  lactic,  oxaHc,  salicylic,  tannic,  and  butyric, 
and  0.2  per  cent  hydrochloric,  sulphuric,  nitric,  arsenious,  and  com- 
bined hydrochloric.  To  each  acid  add  a  few  drops  of  the  glycerol 
extract  of  pig's  stomach  and  a  small  piece  of  fibrin.  Shake  well, 
place  at  40°  C,  and  note  the  progress  of  digestion.  In  which  tubes 
does  the  most  rapid  digestion  occur? 

6.  Influence  of  Metallic  Salts,  etc. — Prepare  a  series  of  tubes 
and  into  each  tube  introduce  4  c.c.  of  pepsin-hydrochloric  acid  solu- 
tion and  I  2  c.c.  of  one  of  the  chemicals  listed  in  Experiment  18 
under  Salivary  Digestion,  page  58.  Introduce  a  small  piece  of  fibrin 
into  each  of  the  tubes  and  keep  them  at  40°  C.  for  one-half  hour. 
Xote  the  variations  in  the  progress  of  digestion.  Where  has  the  least 
rapid  digestion  occurred  ? 

7.  Sahli's  Desmoid  Reaction. — This  is  a  method  for  testing 
gastric  function  without  using  the  stomach  tube.  The  underlying 
principle  of  the  test  is  the  fact  that  raw  catgut  may  be  digested  in 
gastric  juice  but  is  entirely  indigestible  in  pancreatic  juice.  The 
test  is  made  as  follows:  A  mcthyleneblue  pill  is  introduced  into  a  small 
rubber  bag  and  the  mouth  of  the  bag  subsequently  tied  with  catgut.^ 
The  small  bag  is  then  ingested  immediately  after  the  mid-day  meal 
and  the  urine  examined  5,  7,  9  and  18-20  hours  later  for  methylene 
blue.  If  methylene  blue  is  present  in  appreciable  (|uantity,  it  will 
impart  to  the  urine  a  grccnish-blue  color.  If  not  present  in  suflicient 
amount  to  imy)art  this  color  the  urine  should  be  boiled  with  1/5  its 
volume  of  glacial  acetic  acid,  whereupon  a  greenish-blue  color  results 
if  the  chromogen  of  methylene  blue  is  present.  'J'his  contingency 
seldom  arises,  however,  inasmuch  as  in  most  cases  of  uncolored  urine 
it  will  be  found  that  the  ru})l)er  bag  has  passed  through  the  stomach 

'  About  0.05  ^nim  of  mclliylcnc  blue  is  mixed  with  suri'icifiiL  rxl.  f^lyryrrliizrr  lo  form 
a  pill  aV)out  3-4  mm.  in  diameter.  'I'he  pill  is  then  jjlaced  in  the  (enter  of  a  s(|uare  juece  of 
thin  nihbcr  dam  anrl  a  little  baj<-like  receptacle  constructed  by  a  twisting  movement. 
The  neck  of  the  bag  is  then  closed  by  wrapi/ing  three  turns  of  catgut  aiiout  it.  The  most 
satisfactory  catgut  lo  use  is  member  00  nm'  r.alxui  wliich  has  i)revioiisly  liccn  soaked  in 
water  until  Sfjft.  When  ready  for  use  the  l;ag  should  sink  instantly  when  placed  in  water 
and  be  water-tight. 


GASTRIC    DIGESTION.  1 25 

unopened.    It  the  methylene  blue  is  found  in  the  urine  inside  of  18-20 
hours  a  satisfactory  gastric  function  is  indicated. 

8.  Testing  the  Motor  and  Functional  Activities  of  the 
Stomach. — This  test  is  performed  the  same  as  Experiment  19  under 
Salivary  Digestion,  page  58.  If  the  experiment  was  carried  out  under 
salivary  digestion  it  will  not  be  necessary  to  repeat  it  here. 

9.  Influence  of  Bile. — Prepare  five  tubes  as  follows: 

(a)  Five  c.c.  of  pepsin-hydrochloric  acid  solution  +  1/2-1  c.c.  of 
bile. 

(6)  Five  c.c.  of  pepsin-hydrochloric  acid  solution -J- 1-2  c.c.  of 
bile. 

(c)  Five  c.c.  of  pepsin-hydrochloric  acid  solution +  2-3  c.c.  of 
bile. 

(d)  Five  c.c.  of  pepsin-hydrochloric  acid  solution -f  5  c.c.  of  bile. 

(e)  Five  c.c.  of  pepsin-hydrochloric  acid  solution. 

Introduce  into  each  tube  a  small  piece  of  fibrin.  Keep  the  tubes 
at  40°  C.  and  note  the  progress  of  digestion.  Does  the  bile  exert 
any  appreciable  influence  ?    How  ? 

10.  Influence  of  Gastric  Rennin  on  Milk. — Prepare  a  series  of 
five  tubes  as  follows: 

(a)  Five  c.c.  of  fresh  milk -f  0.2  per  cent  hydrochloric  acid  (add 
slowly  until  precipitate  forms). 

(b)  Five  c.c.  of  fresh  milk -f  5  drops  of  rennin  solution. 

(c)  Five  c.c.  of  fresh  milk -f- 10  drops  of  0.5  per  cent  sodium  car- 
bonate solution. 

(d)  Five  c.c.  of  fresh  milk -t- 10  drops  of  a  saturated  solution  of 
ammonium    oxalate. 

(e)  Five  c.c.  of  fresh  milk -f  5  drops  of  0.2  per  cent  hydrochloric 
acid.  Now  to  each  of  the  tubes  (c),  (d),  and  (e)  add  5  drops  of  rennin 
solution.  Place  the  whole  series  of  five  tubes  at  40°  C.  and  after  10-15 
minutes  note  what  is  occurring  in  the  different  tubes.  Give  a  reason 
for  each  particular  result. 

11.  Tests  for  Lactic  Acid. — (a)  Uffelmann's  Reaction. — To  a 
small  quantity  of  Uffelmann's  reagent^  in  a  test-tube  add  a  few  drops 
of  a  lactic  acid  solution.  The  amethyst-blue  color  of  the  reagent  is 
displaced  by  a  straw  yellow.  Other  organic  acids  give  a  similar 
reaction.  Mineral  acids  such  as  hydrochloric  acid  discharge  the  blue 
coloration  leaving  a  colorless  solution. 

{h)  Ferric  Chloride  Test. — Place  10  c.c.  of  very  dilute  ferric  chloride 

'  Uffelmann's  reagent  is  prepared  by  .adding  ferric  chloride  solution  to  a  i  per  cent 
solution  of  carbolic  acid  until  an  amethyst-blue  color  is  obtained. 


12b  PHYSIOLOGICAL    CHEMISTRY. 

in  each  of  live  tubes.  To  the  tirst  add  2  c.c.  of  0.2  per  cent  hydro- 
chloric acid,  to  the  second  2  c.c.  of  10  per  cent  alcohol,  to  the  third 
2  c.c.  of  2  per  cent  sucrose,  to  the  fourth  2  c.c.  of  lactic  acid  and  to  the 
fifth  2  c.c.  of  peptone  solution. 

It  is  evident  from  the  results  obtained  that  neither  of  the  tests 
,^i\en  above  is  satisfactory  for  the  detection  of  lactic  acid  in  the  pres- 
ence of  other  substances  such  as  we  find  in  the  gastric  contents. 

A  satisfactory  deduction  regarding  the  presence  of  lactic  acid  can 
only  be  made  after  extracting  the  gastric  contents  with  ether,  evapor- 
ating the  ether  extract  to  dryness,  and  dissolving  the  residue  in  water. 
This  residue  will  not  contain  any  of  the  contaminations  which  inter- 
fere with  the  simple  tests  as  tried  abo\'e,  and  therefore  if  either  of 
the  tests  is  now  tried  on  the  dissohed  residue  of  the  ether  extract  we 
may  form  an  accurate  conclusion  regarding  the  presence  of  lactic  acid. 

ic)  Hopkins'  Thiophene  Reaction. — Place  about  5  c.c.  of  concen- 
trated sulphuric  acid  in  a  test-tube  and  add  one  drop  of  a  saturated 
solution  of  cupric  sulphate.^  Introduce  a  few  drops  of  the  solution 
to  be  tested,  shake  the  tube  well,  and  immerse  it  in  the  boiling  water 
of  a  beaker-water-bath  for  one  or  two  minutes.  Now  remo\'e  the 
tube,  cool  it  under  running  water,  add  2-3  drops  of  a  dilute  alcoholic 
solution-  of  thiophene,  C^H^S,  from  a  pipette,  replace  the  tube  in 
the  beaker  and  carefully  observe  any  color  change  which  may  occur. 
Lactic  acid  is  indicated  by  the  appearance  of  a  bright  cherry-red  color 
which  forms  rapidly.  This  color  may  be  made  more  or  less  permanent 
by  cooling  the  tube  as  soon  as  the  color  is  produced.  Excess  of  thio- 
phene produces  a  deep  yellow  or  brown  color  with  sulphuric  acid. 
The  test  is  not  wholly  specific  though  the  author  claims  it  to  be  more 
so  than  Uffelmann's  reaction. 

12.  Qualitative  Analysis  of  Stomach  Contents.  Take  100  c.c. 
of  stomach  contents  and  analyze  it  according  to  the  following  scheme: 

'  This  is  adclffl  to  ratalyzc  the  (j.xuliitioii  wliii  li  follnw-s. 
-' Ahout  10-20  flrops  in  loo  c.c.  of  <;5  |)C'r(c-nt  aliohol. 


GASTRIC    DIGESTION. 
Stomach  Contents. 
Filter  and  test  the  fihrate  for  free  hydrochloric  acid. 


12' 


Filtrate  I. 
Divide  into  two  parts. 


Residue. 
Discard    after    making 
amination. 


a    microscopical    ex- 


Filtrate  II. 
One-fifth  portion. 
Test  for: 

(a)  Pepsin. 

(h)  Bile  (see  p. 

(c)  Starch. 

(d)  De.xtrin. 


131). 


Fihrate  III. 
Four-fifths  portion. 

Neutralize  carefully;  any  precipitate  is  acid 
metaprotein  (acid  albuminate).  If  a  precipitate 
forms  filter  and  di\"ide  the  filtrate  into  two  parts. 
If  no  precipitate  forms  divide  the  solution  into 
lico  parts  without  filtering. 

I 


'Filtrate  IV. 
Two-thirds  portion. 
Heat    to    boiling    to    remove    coagulable 
proteins.     If  any  precipitate  forms  filter 
it  off;  if  there  is  no  precipitate  proceed 
directly  with  the  tests. 
Test  for: 

(a)  Sugar.      (DilTerentiate  betvveen  the  various 

sugars  bv  the  use  "of  the  scheme  on  page 
50.) 

(b)  Proteoses. 

(c)  Peptones. 


Filtrate  \'. 
One-thini  portion. 
Test  for: 

(a)  Lactic  acid. 

(b)  Gastric  rennin. 

(c)  Salivary  amylase. 


CHAPTER  VH. 

FATS. 

Fats  occur  very  widely  distributed  in  the  plant  and  animal  king- 
doms, and  constitute  the  third  general  class  of  food  stuffs.  In  plant 
organisms  they  are  to  be  found  in  the  seeds,  roots,  and  fruit  while 
each  individual  tissue  and  organ  of  an  animal  organism  contains 
more  or  less  of  the  substance.  In  the  animal  organism  fats  are  especially 
abundant  in  the  bone  marrow  and  adipose  tissue.  They  contain 
the  same  elements  as  the  carbohydrates,  t.  e.,  carbon,  hydrogen,  and 
oxygen,  but  the  oxygen  is  present  in  smaller  percentage  than  in  the 
carbodydrates  and  the  hydrogen  and  oxygen  are  not  present  in  the 
proportion  to  form  water. 


J-'iG.  35. — Bkkt  ]-".\r.     (Lonj^.) 

Chemically  c(jnsifkre(l  the  fats  are  esters'  of  the  tri  atomic  alcohol, 
glycerol,  and  the  mono-basic  fatty  acids.  In  the  formation  of  these 
fats  three  molecules  of  water  result.  This  water  may  arise  in  either 
of  two  ways.  First,  by  the  replacement  of  the  II  of  each  of  the  Oil 
groups  of  glycerol  by  a  fatty  acid  radical,  giving  the  following  formula 
in  which  R,  R'  and  R"  re|)resent  fatty  acid  radicals, 

'  An  ester  is  an  ethereal  sail  lonsislin^;  of  ;in  or^anii    i,iili(;il  unili-d  wiili  ihe    residue 
of  an  inorganic  or  organic  add. 

128 


FATS,  129 


CH2OR 


CH   OR' 
CH2-OR''. 

Second,  by  the  replacement  of  the  H's  of  the  carboxyl  groups  of  the 
three  fatty  acid  molecules  by  the  glycerol  radical,  thus  yielding  the 
following  type  of  formula  in  which  R  represents  the  glycerol  radical, 

00CH3,C,, 

/ 
R-OOCH3,C,, 

OOCH3,C,, 

Of  these  two  processes  the  second  is  the  more  logical  procedure  from 
the  standpoint  of  the  ionic  theory.  The  three  fatty  acid  radicals 
entering  into  the  structure  of  a  neutral  fat  may  be  the  radicals  of  the 
same  fatty  acid  or  they  may  consist  of  the  radicals  of  three  different 
fatty  acids. 

By  hydrolysis  of  a  neutral  fat,  i.  e.,  by  the  addition  to  the  molecule 
of  those  elements  which  are  eliminated  in  the  formation  of  the  fat 
from  glycerol  and  fatty  acid,  it  may  be  resolved  into  its  component 
parts,  i.  e.,  glycerol  and  fatty  acid.  In  the  case  of  tripalmitin  the  fol- 
lowing would  be  the  reaction: 

C3H,(0-C,,H3,CO)3+3H30  =  C3H,(OH)3  +  3(C,,H3,COOH). 

Tri-palmitin.  Glycerol.  Palmitic  acid. 

This  process  is  called  saponification  and  may  be  produced  by  boiling 
with  alkalis;  by  the  action  of  steam  under  pressure;  by  long-continued 
contact  with  air  and  light;  by  the  action  of  certain  bacteria  and  by 
fat-splitting  enzymes  or  lipases,  e.  g.,  pancreatic  lipase  (see  page  139). 
The  cells  forming  the  walls  of  the  intestines  evidently  possess  the 
peculiar  property  of  synthesizing  the  glycerol  and  fatty  acid  thus 
formed  so  that  after  absorption  these  bodies  appear  in  the  blood  not 
in  their  individual  form  but  as  neutral  fats.  This  synthesis  is  similar 
to  that  enacted  in  the  absorption  of  protein  material  where  the  pep- 
tones are  synthesized  into  albumin  in  the  act  of  absorption. 

The  principal  animal  fats  with  which  we  have  to  deal  are  stearin, 

palmitin,  olein,  and  butyrin.     Such  less  important  forms  as  laurin  and 

myristin  may  occur  abundantly  in  plant  organisms.     The  generally 

accepted  system  of  nomenclature  for  these  fats  is  to  apply  the  prefix 

9 


130  PHYSIOLOGICAL    CHEMISTRY. 

'tri'"  in  each  case  {e.  g.,  /r/-palmitin)  since  three  fatty  acid  radicals 
are  contained  in  the  neutral  fat  molecule. 

Fats  occur  ordinarily  as  mixtures  of  se\cral  individual  fats.  For 
example,  the  fat  found  in  animal  tissues  is  a  mixture  of  tri-olein,  tri- 
palmitin  and  tri-stearin,  the  percentage  of  any  one  of  these  fats  present 
depending  upon  the  particular  species  of  animal  from  whose  tissue 
the  fat  was  derived.  Thus  the  ordinary  mutton  fat  contains  more  tri- 
stearin  and  less  tri-olein  than  the  pork  fat.  Human  fat  contains  from 
67  per  cent  to  85  per  cent  of  tri-olein  and  according  to  Benedict  and 
Osterberg,  upon  analysis  yields  76.08  per  cent  of  carbon  and  11.78 
per  cent  of  hydrogen. 

Pure  neutral  fats  are  odorless,  tasteless,  and  generally  colorless. 
They  are  insoluble  in  the  ordinary  protein  soh'ents  such  as  water, 
salt  solutions,  and  dilute  acids  and  alkalis,  but  are  very  readily  soluble 
in  ether,  benzene,  chloroform,  and  boiling  alcohol.  The  neutral  fats 
are  non- volatile  substances  possessing  a  neutral  reaction.  If  allowed 
to  remain  in  contact  with  the  air  for  a  sufficient  length  of  time  they 
become  yellow  in  color,  assume  an  acid  reaction  and  are  said  to  be 
rancid.  The  neutral  fats  may  be  crystallized,  some  of  them  with  great 
facility.  The  crystalline  forms  of  some  of  the  more  common  fats  are 
reproduced  in  Figs.  35,  36  and  37  on  pages  128,  131  and  133.  Each 
individual  fat  possesses  a  specific  melting-  or  boiling-point  (according 
to  whether  the  body  is  solid  or  fluid  in  character)  and  this  property 
of  melting  or  boiling  at  a  definite  temperature  may  be  used  as  a  means 
of  difTerentiation  in  the  same  way  as  the  coagulation  temperature  (see 
page  1 09)  is  used  for  the  differentiation  of  coagulable  proteins.  When 
shaken  with  water,  or  a  solution  of  albumin,  soap,  or  acacia,  the  liquid 
fats  are  finely  divided  and  assume  a  condition  known  as  an  emulsion. 
The  emulsion  with  water  is  transitory,  while  Ihc  emulsions  with  soa]), 
acacia,  or  albumin,  are  permanent. 

The  fat  ingested  continues  essentially  unaltered  until  it  reaches 
the  intestines  where  it  is  acted  upon  by  pancreatic  lipase  {steapsin)  the 
fat-splitting  enzyme  of  the  pancreatic  juice  (see  page  139),  and  glycerol 
and  fatty  acid  are  formed  from  a  large  portion  of  the  fat.  Part  of  the 
fatty  acid  thus  formed  is  dissolved  in  the  bile  and  absorbed  while  the 
remainder  unites  with  the  alkalis  of  the  pancreatic  juice  and  forms 
soluble  soaps,  'i'hese  soaps  may  further  act  to  j)roduce  an  emulsion 
of  the  remaining  fat  and  thus  aid  in  its  absorj)tion.  'J'hat  bile  is  of 
assistance  in  the  absorption  of  fat  is  indicated  by  the  increase  of  fat 
in  the  feces  when  for  any  reason  bile  does  not  pass  into  the  intestines. 
'I'hat  fa-t  is  not  absorbed  unsi)lit  in  the  form  of  an  emulsion  has  recently 


FATS.  131 

been  redemonstrated  by  Whitehead^  in   a   histological  study  of  the 
absorption  of  fat  stained  with  Sudan  III. 

The  fat  distributed  throughout  the  animal  body  is  formed  partly 
from  the  ingested  fat  and  partly  from  carbohydrates  and  the  "carbon 
moiety"  of  protein  material.  The  formation  of  adipocere  and  the 
occurrence  of  fatty  degeneration  are  sometimes  given  as  proofs  of  the 
formation  of  fat  from  protein.  This  is  questioned  by  many  investi- 
gators. Rather  more  satisfactory  and  direct  proof  of  the  formation 
of  fat  from  protein  material  has  been  obtained  by  Hofmann  in  experi- 


MuTTON  Fat.     (Long.) 


mentation  with  fly-maggots.  The  normal  content  of  fat  in  a  number 
of  maggots  was  determined  and  later  the  fat  content  of  others  which 
had  developed  in  blood  (84  per  cent  of  the  solid  matter  of  blood  plasma 
is  protein  material)  was  determined.  The  fat  content  was  found  to 
have  increased  700  to  iioo  per  cent  as  a  result  of  the  diet  of  blood 
proteins.  The  celebrated  experiments  of  Pettenkofer  and  Voit,  how- 
ever, have  furnished  what  is,  perhaps,  the  most  substantial  positive 
evidence  of  the  formation  of  fat  from  protein.  These  investigators 
fed  dogs  large  amounts  of  lean  meat,  daily,  and  through  subsequent 
urinary  and  fecal  examinations  were  enabled  to  account  for  o-nly  part 
of  the  ingested  carbon,  although  obtaining  a  satisfactory  nitrogen 
balance.  The  discrepancy  in  the  carbon  balance  was  explained  upon 
the  theory  that  the  protein  of  the  ingested  meat  had  been  split  into 
a  nitrogenous  and  a  non-niirogenous  portion  in  the  organism,  and  that 
the  non-nitrogenous  portion,  the  so-called  "carbon  moiety"  of  the 
protein,  had  been  subsequently  transformed  into  fat  and  deposited 

'  Wliitehead:     American  Journal  of  Physiology,  X.'S.Y,  igio.     Proceedings  n.  28. 


132  PHYSIOLOGICAL    CHEMISTRY. 

as  such  in  the  tissues  of  the  organism.  Some  investigators  are  not 
inclined  to  accept  these  data  regarding  the  formation  of  fat  from 
protein  as  conclusive. 

The  latest  evidence  in  favor  of  the  formation  of  fat  from  protein 
is  furnished  by  the  very  recently  reported  experiments  of  Weinland. 
This  investigator  worked  with  the  larvae  of  CallipJwra^  these  larvae 
being  rubbed  up  in  a  mortar-  with  Witte's  peptone  and  water  to  form 
a  homogeneous  mixture.  After  placing  these  mixtures  at  38°  C.  for 
24  hours  the  fat  content  was  found  to  have  increased,  as  much  as  140 
per  cent  in  some  instances.  The  active  agency  in  this  transformation 
of  fat  is  the  larval  tissue  since  the  tissues  of  both  the  dead  and  living 
larvae  possess  the  property.  Data  are  given  from  control  tests  which 
show  that  the  action  of  bacteria  in  this  transformation  of  protein  was 
excluded. 


Experiments  on  Fats. 

1.  Solubility. — Test  the  solubility  of  olive  oil  in  each  of  the 
ordinary  solvents  (see  page  22)  and  in  cold  alcohol,  hot  alcohol,  chloro- 
form, ether,  and  carbon  tetrachloride. 

2.  Formation  of  a  Transparent  Spot  on  Paper. — Place  a  drop 
of  olive  oil  upon  a  piece  of  ordinary  writing  paper.  Note  the  trans- 
parent appearance  of  the  paper  at  the  point  of  contact  with  the  fat. 

3.  Reaction. — Try  the  reaction  of  fresh  olive  oil  to  litmus.  Repeat 
the  test  with  rancid  oli\c  oil.  What  is  the  reaction  of  a  fresh  fat  and 
how  does  this  reaction  change  upon  allowing  the  fat  to  stand  for 
some   time  ? 

4.  Formation  of  Acrolein. — To  a  little  olive  oil  in  a  mortar 
add  some  dry  potassium  bisulphate,  KHSO^,  and  rub  u])  thoroughly. 
Transfer  to  a  dry  test-tube  and  cautiously  heat.  Note  the  irritating 
odor  of  acrolein.  The  glycerol  of  the  fat  has  been  dchydrolyzed  and 
acrylic  aldehyde  or  acrolein  has  been  produced.  This  is  the  reaction 
which  takes  place: 

CH^OH  CHO 

I  I 

CHOH      —     CH-f2H20. 

CH^OH  CH^ 

Glycerol.  Acrolein, 

'  The  ordinary  "  blow-fly." 

*  Intact  larva;  were  used  in  .some  experiments. 


FATS. 


^33 


5.  Emulsification. — (a)  Shake  up  a  drop  of  neutral^  olive  oil 
with  a  little  water  in  a  test-tube.  The  fat  becomes  finely  divided, 
forming  an  emulsion.  This  is  not  a  permanent  emulsion  since  the 
fat  separates  and  rises  to  the  top  upon  standing. 

(b)  To  5  c.c.  of  water  in  a  test-tube  add  2  or  3  drops  of  0.5  per 
cent  NajCOg.  Introduce  into  this  faintly  alkaline  solution  a  drop 
of  neutral  olive  oil  and  shake.  The  emulsion  while  not  permanent 
is  not  so  transitory  as  in  the  case  of  water  free  from  sodium  carbonate. 

(c)  Repeat  (b)  using  rancid  olive  oil.  What  sort  of  an  emulsion 
do  you  get  and  why  ? 

(d)  Shake  a  drop  of  neutral  olive  oil  with  dilute  albumin  solution. 
What  is  the  nature  of  this  emulsion  ?    Examine  it  under  the  microscope. 

6.  Fat  Crystals. — Dissolve  a  small  piece  of  lard  in  ether  in  a 
test-tube,   add  an  equal  volume  of  alcohol  and  allow  the  alcohol- 


FiG.  37. — Pork  Fat. 


ether  mixture  to  evaporate  spontaneously.  Examine  the  crystals  under 
the  microscope  and  compare  them  with  those  reproduced  in  Figs. 
35,  36  and  37,  on  pages  128,  131  and  133. 

7.  Saponification  of  Bayberry  Tallow.- — Fill  a  large  casserole 
two-thirds  full  of  water  rendered  strongly  alkaline  with  solid  potas- 
sium hydroxide  (a  stick  one  inch  in  length).  Add  about  10  grams 
of  bayberry  tallow  and  boil,  keeping  the  volume  constant  by  adding 

^  Neutral  olive  oil  may  be  prepared  by  shaking  ordinar}'  olive  oil  with  a  ic  per  cent 
solution  of  sodium  carbonate.  This  mixture  should  then  be  extracted  with  ether  and 
the  ether  removed  by  evaporation.     The  residue  is  neutral  olive  oil. 

-  Bayberr}'  tallow  is  derived  from  the  fatty  covering  of  the  berries  of  the  -wax  myrtle. 
It  is  therefore  frequently  called  "myrtle  wax"  or  "bayberry  wax." 


134 


PHYSIOLOGICAL    CHEMISTRY. 


water  as  needed.  When  saponification  is  complete^  remove  25  c.c. 
of  the  soap  sohition  for  use  in  Experiment  8  and  add  concentrated 
hydrochloric  acid  slowly  to  the  remainder  until  no  further  precipitate 
is  produced.-  Cool  the  solution  and  the  precipitate  of  free  fatty  acid 
will  rise  to  the  surface  and  form  a  cake.  In  this  instance  the  fatty 
acid  is  principally  palmitic  acid.  Remove  the  cake,  break  it  into 
small  pieces,  wash  it  with  water  by  decantation  and  transfer  to  a 
small  beaker  by  means  of  95  per  cent  alcohol.  Heat  on  a  water-bath 
until   the  palmitic  acid  is  dissolved,   then    filter  through   a   dry  filter 


Fig.  ,^8. — Palmitic  Acid. 

paper  and  allow  the  filtrate  to  cool  slowly  in  order  to  obtain  satis- 
factory crystals.  Write  the  reactions  which  have  taken  place  in  this 
experiment. 

When  the  palmitic  acid  has  completely  crystallized  filter  off  the 
alcohol,  dry  the  crystals  between  filter  papers  and  try  the  tests  given 
in  Experiment  9,  below. 

8.  Salting-out  Experiment. — To  25  c.c.  of  soap  solution,  pre- 
pared as  described  above,  add  solid  sodium  chloride  to  the  point  of 
saturation,  with  continual  stirring.  A  menstruum  is  thus  formed  in 
which  the  soap  is  insoluble.  This  salting-out  ])rocess  is  entirely 
analogous  to  the  salting-out  oi  ]jroteins  (see  page  94J. 

9.  Palmitic  Acid,  (a)  Examine  the  crystals  under  the  micro- 
scope and  compare  them  with  those  shown  in  Fig.  38,  above. 

'  Place  2  or  T,  drops  in  a  tesl-tuhc  full  of  water.  If  saponilu  :ilioii  is  (:oiii|)lele  the 
prorlucts  will  remain  in  solution  anfl  no  oil  will  se|)ar;Uc. 

^  Unclcr  some  (onrlilions  a  ]>urer  pnxliK  1  is  ohiaim-d  if  the  so.-ip  solution  is  cooled 
before  precipitating  the  fatty  acid. 


FATS. 


135 


oe 


QH^ 


{b)  Solubility. — Try  the  solubility  of  palmitic  acid  in   the  same 
solvents  as  used  on  fats  (see  page  132). 

(c)  Melting-point. — Determine  the  melting-point  of  palmitic  acid 
by  one  of  the  methods  given  on  page  136. 

{d)  Formation  of  Transparent  Spot  on 
Paper. — Melt  a  little  of  the  fatty  acid 
and  allow  a  drop  to  fall  upon  a  piece 
of  ordinary  writing  paper.  How  does 
this  compare  with  the  action  of  a  fat 
under  similar  circumstances  ? 

(e)  Acrolein  Test. — Apply  the  test  as 
given  under  4,  page  132.  Explain  the 
result. 

10.  Saponification  of  Lard. — To 
25  grams  of  lard  in  a  flask  add  75  c.c. 
of  alcoholic-potash  solution  and  warm 
upon  a  water-bath  until  saponification  is 
complete.  (This  point  is  indicated  by 
the  complete  solubility  of  a  drop  of  the 
solution  when  allowed  to  fall  into  a  little 
water.)  Now  transfer  the  solution  from 
the  flask  to  an  evaporating  dish  con- 
taining about  100  c.c.  of  water  and 
heat  on  a  water-bath  until  all  the  alcohol 
has  been  driven  off.  Precipitate  the 
fatty  acid  with  hydrochloric  acid  and 
cool  the  solution.  Remove  the  fatty  acid 
which  rises  to  the  surface,  neutralize 
the  solution  with  sodium  carbonate  and 
evaporate  to  dryness.  Extract  the  residue 
with  alcohol,  remove  the  alcohol  by  evaporation  upon  a  water-bath  and 
on  the  residue  of  glycerol  thus  obtained  make  the  tests  as  given  below. 

11.  Glycerol,   (a)   Taste. — What  is  the  taste  of  glycerol? 

(b)  Solubility. — Try  the  solubility  of  glycerol  in  water,  alcohol 
and  ether. 

(c)  Acrolein  Test. — Repeat  the  test  as  given  under  4,  page  132. 

(d)  Borax  Fusion  Test. — Fuse  a  little  glycerol  on  a  platinum 
wire  with  some  powdered  borax  and  note  the  characteristic  green 
flame.    This  color  is  due  to  the  glycerol  ester  of  boric  acid. 

(e)  Fehling's  Test. — How  does  this  result  compare  with  the 
results  on  the  susjars  ? 


Fig. 


39. — Melting-Poixt   App.4- 

RATUS. 


136  PHYSIOLOGICAL    CHEMISTRY. 

•  (/)  Solutiaii  of  CiuOH)^. — Form  a  little  cupric  hydroxide  by 
mixing  cupric  sulphate  and  potassium  hydroxide.  Add  a  little  glycerol 
to  this  suspended  precipitate  and  note  what  occurs. 

12.  Melting-Point  of  Fat.  First  Method. — Insert  one  of  the 
melting-point  tubes,  furnished  by  the  instructor,  into  the  liquid  fat 
and  draw  up  the  fat  until  the  bulb  of  the  tube  is  about  one-half  full 
of  the  material.  Then  fuse  one  end  of  the  tube  in  the  flame  of  a  bun- 
sen  burner  and  fasten  the  tube  to  a  thermometer  by  means  of  a  rubber 
band  in  such  a  manner  that  the  bottom  of  the  fat  column  is  on  a  level 
with  the  bulb  of  the  thermometer  (Fig.  39,  page  135).  Fill  a  beaker 
of  medium  size  about  two-thirds  full  of  water  and  place  it  within  a 
second  larger  beaker  which  also  contains  water,  the  two  vessels  being 
separated  by  pieces  of  cork.  Immerse  the  bulb  of  the  thermometer  and 
the  attached  tube  in  such  a  way  that  the  bulb  is  about  midway  between 
the  upper  and  the  lower  surfaces  of  the  water  of  the  inner  beaker. 
The  upper  end  of  the  tube  being  open  it  must  extend  above  the  sur- 
face of  the  surrounding  water.  Apply  gentle  heat,  stir  the  water,  and 
note  the  temperature  at  which  the  fat  first  begins  to  melt.  This  point 
is  indicated  by  the  initial  transparency.  For  ordinary  fats,  raise  the 
temperature  \'ery  cautiously  from  30°  C.  To  determine  the  congealing- 
point  remove  the  flame  and  note  the  temperature  at  which  the  fat 
begins  to  solidify.  Record  the  melting-  and  congealing-points  of  the 
various  fats  submitted  by  the  instructor. 

Second  Method. — Fill  a  small  evaporating  dish  about  one-half 
full  of  mercury  and  place  it  on  a  water-bath.  Put  a  small  drop  of  the 
fa,t  under  examination  on  an  ordinary  coverglass  and  place  this  upon 
the  surface  of  the  mercury.  Raise  the  temperature  of  the  water-bath 
slowly  and  by  means  of  a  thermometer  whose  bulb  is  immersed  in  the 
mercury,  note  the  melting-point  of  the  fat.  Determine  the  congealing- 
point  by  removing  the  flame  and  leaving  the  fat  drop  and  coverglass 
in  position  upon  the  mercury.  How  do  the  melting-points  as  deter- 
mined by  this  method  compare  with  those  as  determined  by  the  first 
method?     Which  method  is  the  more  accurate,  and  why? 


CHAPTER  VIII. 
PANCREATIC  DIGESTION. 

As  soon  as  the  food  mixture  leaves  the  stomach  it  comes  into 
intimate  contact  with  the  bile  and  the  pancreatic  juice.  Since  these 
fluids  are  alkaline  in  reaction  there  can  obviously  be  no  further  peptic 
activity  after  they  have  become  intimately  mixed  with  the  chyme  and 
have  neutralized  the  acidity  previously  imparted  to  it  by  the  hydro- 
chloric acid  of  the  gastric  juice.  The  pancreatic  juice  reaches  the 
intestine  through  the  duct  of  Wirsung  which  opens  into  the  intestine 
near  the  pylorus. 

Normally  the  secretion  of  pancreatic  juice  is  brought  about  by  the 
stimulation  produced  by  the  acid  chyme  as  it  enters  the  duodenum. 
This  secretion  is  probably  not  due  to  a  nervous  reflex  as  was  believed 
by  Pawlow  but  rather,  as  Bayhss  and  Starling  have  shown,  is  depend- 
ent upon  the  presence,  in  the  epithelial  cells  of  the  duodenum  and 
jejunum  of  a  body  known  as  prosecretin.  This  body  is  changed  into 
secretin}  through  the  hydrolytic  action  of  the  acid  present  in  the  chyme. 
The  secretin  is  then  absorbed  by  the  blood,  passes  to  the  pancreas 
and  stimulates  the  pancreatic  cells,  causing  a  flow  of  pancreatic  juice. 
The  quantity  of  juice  secreted  under  these  conditions  is  proportional 
to  the  amount  of  secretin  present.  The  activity  of  secretin  solutions 
is  not  diminished  by  boiling,  hence  the  body  does  not  react  like  an 
enzyme.  Further  study  of  the  body  may  show  it  to  be  a  definite 
chemical  individual  of  relatively  low  molecular  weight.  It  has  not 
been  possible  thus  far  to  obtain  secretin  from  any  tissues  except  the 
mucous  membrane  of  the  duodenum  and  jejunum. 

The  juice  as  obtained  from  a  permanent  fistula  differs  greatly  in 
its  properties  from  the  juice  as  obtained  from  a  temporary  fistula, 
and  neither  form  of  fluid  possesses  the  properties  of  the  normal  fluid. 
Pancreatic  juice  collected  by  Glaessner  from  a  natural  fistula  has 
been  found  to  be  a  colorless,  clear,  strongly  alkaline  fluid  which  foams 
readily.  It  is  further  characterized  by  containing  albumin,  globulin, 
proteose,  and  peptone;  nucleoprotein  is  also  present  in  traces.^  The 
average  daily  secretion  of  pancreatic  juice  is  650  c.c.  and  its  specific 

^  Secretin  belongs  to  the  class  of  substances  called  hormones  or  chemical  messengers. 
-  Glaessner:     Zeitschrift  fur  physiulogische  Chemie,  1Q04,  40,  p.  476. 


138  PHYSIOLOGICAL    CHEMISTRY. 

gravity  is  1.008.  The  fluid  contains  1.3  per  cent  of  solid  matter  and 
the  freezing-point  is  — 0.47°  C.  The  normal  pancreatic  secretion 
contains  at  least  four  distinct  enzymes.  They  are  trypsin,  a  proteolytic 
enzyme;  pancreatic  amylase  (amylopsin),  an  amylolytic  enzyme; 
pancreatic  lipase  (steapsin),  a  fat-splitting  enzyme;  and  pancreatic 
rennin,  a  milk-coagulating  enzyme.  Lactase,  the  lactose-splitting 
enzyme,  is  also  present  at  certain  times. 

The  most  important  of  the  four  enzymes  of  the  pancreatic  juice  is 
the  proteolytic  enzyme  trypsin.  This  enzyme  resemljles  pepsin  in  so 
far  as  each  has  the  power  of  breaking  down  protein  material,  but 
the  trypsin  has  much  greater  digestive  power  and  is  able  to  cause  a 
more  complete  decomposition  of  the  complex  protein  molecule.  In 
the  process  of  normal  digestion  the  protein  constituents  of  the  diet 
are  for  the  most  part  transformed  into  proteoses  (albumoses)  and 
peptones  before  coming  in  contact  with  the  enzyme  trypsin.  This  is 
not  absolutely  essential  however,  since  trypsin  possesses  digestive 
activity  sufficient  to  transform  unaltered  native  proteins  and  to  pro- 
duce from  their  complex  molecules  comparatively  simple  fragments. 
Among  the  products  of  tryptic  digestion  are  proteoses,  peptones,  pep- 
tides, leucine,  tyrosine,  aspartic  acid,  glutamic  acid,  alanine,  phenylal- 
anine, glycocoll,  cystine,  serine,  valine,  proline,  oxyproline,  isoleucine, 
arginine,  lysine,  histidine,  and  tryptophane.  (The  crystalline  forms 
of  many  of  these  products  are  reproduced  in  Chapter  1\'.)  Trypsin 
does  not  occur  preformed  in  the  gland,  but  exists  there  as  a  zymogen 
called  trypsinogen  which  bears  the  same  relation  to  trypsin  that  pep- 
sinogen does  to  pepsin.  Trypsin  has  never  been  obtained  in  a  pure 
form  and  therefore  very  little  can  be  stated  definitely  as  to  its  nature. 
The  enzyme  is  the  most  active  in  alkaline  solution  but  is  also  active 
in  neutral  or  slightly  acid  solutions.  Trypsin  is  destroyed  by  mineral 
acids  and  may  also  be  destroyed  by  comparatively  weak  alkali  (2  per 
cent  sodium  carbonate)  if  left  in  contact  for  a  sufficiently  long  time. 
Trypsinogen,  on  the  other  hand,  is  more  resistant  to  the  action  of 
alkalis.  In  j>ancreatic  dij^^eslion  the  protein  does  not  swell  as  is  the 
case  in  gastric  digestion,  but  becomes  more  or  less  "honeycombed" 
and  it  finally  disintegrates. 

The  pancreatic  juice  which  is  collected  by  means  of  a  fistula 
possesses  practically  no  power  to  digest  protein  matter.  A.  body 
called  enterokinase  occurs  in  the  intestinal  juice  and  has  the  power 
of  converting  trypsinogen  into  tryj)sin.  'J'his  ])r()cess  is  known  as  the 
''activation"  of  tryj)sinogen  and  through  it  a  juice  which  is  incapable 
(){  digesting  protein  may  be  made  active.     Enterokinase  is  not  always 


PANCREATIC    DIGESTION.  1 39 

present  iii  the  intestinal  juice  since  it  is  secreted  only  after  the  pan- 
creatic juice  reaches  the  intestine.  It  resembles  the  enzymes  in  that 
its  activity  is  destroyed  by  heat,  but  differs  materially  from  this  class 
of  bodies  in  that  a  certain  quantity  is  capable  of  activating  only  a 
definite  quantity  of  trypsinogen.  It  is,  however,  generally  classified 
as  an  enzyme.  Enterokinase  has  been  detected  in  the  higher  animals, 
and  a  kinase  possessing  similar  properties  has  been  shown  to  be 
present  in  bacteria,  fungi,  impure  fibrin,  lymph  glands,  and  snake- 
venom.  The  activation  of  trypsinogen  into  trypsin  may  be  brought 
about  in  the  gland  as  well  as  in  the  intestine  of  the  living  organism 
(Mendel  and  Rettger).  The  manner  of  the  activation  in  the  gland 
and  the  nature  of  the  body  causing  it  are  unknown  at  present. 

Delezenne  claims  that  trypsinogen  may  be  activated  by  soluble 
calcium  salts.  He  reports  experiments  which  indicate  that  proteo- 
lytically  inactive  pancreatic  juice,  obtained  directly  from  the  duct, 
when  treated  with  salts  of  this  character,  assumes  the  property  of 
digesting  protein  material.  This  process  by  which  the  trypsinogen 
is  activated  through  the  instrumentality  of  calcium  salts  is  very  rapid 
and  is  designated  by  Delezenne  as  an  "explosion."  The  recent  sugges- 
tion of  Mays  that  there  may  possibly  be  several  precursors  of  trypsin 
one  of  which  is  activated  by  enterokinase  and  the  others  by  other 
agents,  is  of  interest  in  this  connection. 

Pancreatic  amylase  {amylopsin),  the  second  of  the  pancreatic 
enzymes,  is  an  amylolytic  enzyme  which  possesses  somewhat  greater 
digestive  power  than  the  salivary  amylase  (ptyalin)  of  the  saliva. 
As  its  name  implies,  its  activity  is  confined  to  the  starches,  and  the 
products  of  its  amylolytic  action  are  dextrins  and  sugars.  The  sugars 
are  principally  iso-maltose  and  maltose  and  these  by  the  further 
action  of  an  inverting  enzyme  are  partly  transformed  into  dextrose. 

It  is  possible  that  the  saliva  as  a  digestive  fluid  is  not  absolutely 
essential.  The  salivary  amylase  (ptyalin)  is  destroyed  by  the  hydro- 
chloric acid  of  the  gastric  juice  and  is  therefore  inactive  when  the 
chyme  reaches  the  intestine.  Should  undigested  starch  be  present 
at  this  point  however,  it  would  be  quickly  transformed  by  the  active 
pancreatic  amylase.  This  enzyme  is  not  present  in  the  pancreatic 
juice  of  infants  during  the  first  few  weeks  of  life,  thus  showing  very 
clearly  that  a  starchy  diet  is  not  normal  for  this  period. 

It  has  been  claimed  that  pancreatic  amylase  has  a  slight  digestive 
action  upon  unboiled  starch. 

The  third  enzyme  of  the  pancreatic  juice  is  called  pancreatic  lipase 
(steapsin)  and  is  a  fat-splitting  enzyme.     It  has  the  power  of  splitting 


I40  PHYSIOLOGICAL    CHEMISTRY. 

the  neutral  fats  of  the  food  by  hydrolysis,  into  fatty  acid  and  glycerol.    A 
typical  reaction  would  be  as  follows: 

C3H,(OCi5H3,CO)3  +  3H,0  =  3(C,,H3jCOOH)  +  C3H5(OH)3. 

Tri-palmitin.  Palmitic  acid.  Glycerol. 

Recent  researches  make  it  probable  that  fats  undergo  saponifica- 
tion to  a  certain  extent  prior  to  their  absorption.  The  fatty  acids 
formed,  in  part  unite  with  the  alkalis  of  the  pancreatic  juice  ,and  in- 
testinal secretion  to  form  soluble  soaps;  in  part  they  are  doubtless 
absorbed  dissolved  in  the  bile.  Some  observers  believe  that  the  fats 
may  also  be  absorbed  in  emulsion — a  condition  promoted  by  the 
presence  of  the  soluble  soaps.  After  absorption  the  fatty  acids  are 
re-synthesized  to  form  neutral  fats  with  glycerol. 

Pancreatic  lipase  is  very  unstable  and  is  easily  rendered  inert 
by  the  action  of  acid.  For  this  reason  it  is  not  possible  to  prepare 
an  extract  having  a  satisfactory  fat-splitting  power  from  a  pancreas 
which  has  been  removed  from  the  organism  for  a  sufficiently  long 
time  to  have  become  acid  in  reaction. 

The  fourth  enzyme  of  the  pancreatic  juice  is  called  pancreatic 
rennin.  It  is  a  milk-coagulating  enzyme  whose  action  is  very  similar 
to  that  of  the  enzyme  gastric  rennin  found  in  the  gastric  juice.  It  is 
supposed  to  show  its  greatest  activity  at  a  temperature  varying  from 
60°  to  65°  C. 


The  enzymes  of  the  intestinal  juice  are  of  great  importance  to 
the  animal  organism.  These  enzymes  include  erepsin  (ercpsase), 
sucrase,  maltase,  lactase,  and  enterokinase. 

Erepsin  is  a  proteolytic  enzyme  which  has  the  property  of  acting 
upon  the  yjroteoses  and  peptones  which  are  formed  through  the 
action  of  trypsin  and  further  splitting  them  into  amino  acids.  Erepsin 
has  no  power  of  digesting  any  native  proteins  cxcej)!  caseinogen, 
histoncs,  and  y^rotamines.  It  possesses  its  greatest  activity  in  an 
alkaline  solution  although  it  is  slightly  active  in  acid  solution.  An 
extract  of  the  intestinal  erepsin  may  be  prepared  by  treating  the  finely 
divided  intestine  of  a  cat,  dog,  or  pig  with  toluene-  or  chloroform-water 
and  permitting  the  mixture  to  stand  with  occasional  shaking  for  24-72 
hours.*  Enzymes  similar  to  erepsin  occur  in  various  tissues  of  the 
organism. 

The  three  invertases  sucrase,  maltase,  and  lactase  are  also  important 
enzymes  of  the  intestinal   mucosa.     The  sucrase  acts  u])on  sucrose 

'  See  ]).  1  2. 


PANCREATIC    DIGESTION.  I4I 

and  inverts  it  with  the  formation  of  invert  sugar  (dextrose  and  laevulose). 
Some  investigators  claim  that  sucrase  is  also  present  in  saliva  and 
gastric  juice.  It  probably  does  not  exist  normally  in  either  of  these 
digestive  juices,  however,  and  if  found  owes  its  presence  to  the  excre- 
tory processes  of  certain  bacteria.  Sucrases  may  also  be  obtained 
from  several  vegetable  sources.  For  investigational  purposes  it  is 
ordinarily  obtained  from  yeast  (see  p.  ii).  It  exhibits  its  greatest 
activity  in  the  presence  of  a  slight  acidity  but  if  the  acidity  be  increased 
to  any  extent  the  reaction  is  inhibited. 

Lactase  is  an  enzyme  which  inverts  lactose  with  the  consequent 
formation  of  dextrose  and  galactose.  Its  action  is  entirely  analogous, 
in  type,  to  that  of  sucrase.  It  has  apparently  been  proven  that  lactase 
occurs  in  the  intestinal  mucosa  of  the  young  of  all  animals  which 
suckle  their  offspring.^  It  may  also  occur  in  the  intestinal  mucosa 
of  certain  adult  animals  if  such  animals  be  maintained  upon  a  ration 
containing  more  or  less  lactose.  Fischer  and  Armstrong  have  demon- 
strated the  reversible  action^  of  lactase. 

For  discussions  of  maltase  and  enterokinase  see  pages  54  and  138 
respectively. 

PREPARATION  OF  AN  ARTIFICIAL  PANCREATIC  JUICE.^ 

After  removing  the  fat  from  the  pancreas  of  a  pig  or  sheep,  finely 
divide  the  organ  by  means  of  scissors  and  grind  it  in  a  mortar.  If 
convenient,  the  use  of  an  ordinary  meat  chopper  is  a  very  satisfactory 
means  of  preparing  the  pancreas. 

When  finely  divided  as  above  the  pancreas  should  be  placed  in  a 
500  c.c.  flask,  about  150  c.c.  of  30  per  cent  alcohol  added  and  the 
flask  and  contents  shaken  frequently  for  twenty-four  hours.  (What 
is  the  reaction  of  this  alcoholic  extract  at  the  end  of  this  period,  and 
why?)  Strain  the  alcoholic  extract  through  cheese  cloth,  filter,  nearly 
neutralize  with  potassium  hydroxide  solution  and  then  exactly  neutral- 
ize it  with  0.5  per  cent  sodium  carbonate. 

Products  of  Tryptic  Digestion. 

Take  about  200  grams  of  lean  beef  which  has  been  freed  from  fat 
and  finely  ground  and  place  it  in  a  large-sized  beaker.  Introduce 
equal  volumes  of  the  pancreatic  extract  prepared  as  above  and  0.5 

'Mendel  and  Mitchell:  American  Journal  of  Physiology,  1907,  XX,  p.  Si. 
-  See  p.  6. 

^For  other  methods  of  preparation  see  Karl  Mays:  Zeitschrift  fiir  pJiysiohgische 
Ckemie,  1903,  XXXVIII,  p.  428. 


142  PHYSIOLOGICAL    CHEMISTRY. 

per  cent  sodium  carbonate,  add  5  c.c.  of  an  alcoholic  solution  of  thymol 
to  prevent  putrefaction,  and  place  the  beaker  in  an  incubator  at  40°  C. 
Stir  the  contents  of  the  beaker  frequently  and  add  more  thymol  if  it 
becomes  necessary.  Allow  digestion  to  proceed  for  from  2  to  5  days 
and  then  separate  the  products  formed  as  follows:  Strain  off  the  undis- 
solved residue  through  cheese  cloth,  nearly  neutralize  the  solution 
with  dilute  hydrochloric  acid  and  then  exactly  neutralize  it  with  0.2  per 
cent  hydrochloric  acid.  A  precipitate  at  this  point  would  indicate 
alkali  meta protein  (alkali  albuminate).  Filter  off  any  precipitate  and 
divide  the  filtrate  into  two  parts,  a  one-fourth  and  a  three-fourth 
portion. 

Transfer  the  one-fourth  portion  to  an  evaporating  dish  and  make 
the  separation  of  proteoses  and  peptones  as  well  as  the  final  tests  upon 
these  bodies  according  to  the  directions  given  on  page  no. 

Place  about  5  c.c.  of  the  three-fourth  portion  in  a  test-tube  and 
add  about  i  c.c.  of  bromine  water.  A  violet  coloration  indicates  the 
presence  of  tryptophane  (see  page  72).  Concentrate^  the  remainder 
of  the  three-fourth  portion  to  a  thin  syrup  and  make  the  separation  of 
leucine  and  tyrosine  according  to  the  directions  given  on  page  80. 


GENERAL  EXPERIMENTS  ON  PANCREATIC  DIGESTION. 

Experiments  on  Trypsin. 

I.  The  Most  Favorable  Reaction  for  Tryptic  Digestion. — 
Prepare  seven  tubes  as  follows: 

(a)  2-3  c.c.  of  neutral  pancreatic  extract  +  2-3  c.c.  of  water. 

{b)  2-3  c.c.  of  neutral  pancreatic  extract  -f-  2-3  c.c.  of  i  per  cent 
sodium  carbonate. 

ic)  2-3  c.c.  of  neutral  pancreatic  extract  +2-3  c.c.  of  0.5  per  cent 
sodium  carbonate. 

{d)  2-3  c.c.  of  neutral  pancreatic  extract  4-2-3  c.c.  of  0.2  per  cent 
hydrochloric  acid. 

(e)  2-3  c.c.  of  neutral  pancreatic  extract  4-2-3  c.c.  of  0.2  yier  cent 
rnmbined  hydrochloric  acid. 

(/)  2-3  c.c.  of  neutral  pancreatic  extract  +2-3  c.c.  of  0.4  per  cent 
boric  acid. 

ig)  2-3  c.c.  of  neutral  jjancreatic  extract  +2-3  c.c.  of  0.4  per  cent 
acetic  acid. 

'  If  the  solution  is  alkaline  in  reaction,  uliile  it  is  bein^  concciitialcd,  lli<-  amino  acids 
will  be  broken  flown  anrl  ammonia  will  be  liberated. 


PANCREATIC    DIGESTION.  I43 

Add  a  small  piece  of  fibrin  to  the  contents  of  each  tube  and  keep 
them  at  40°  C.  noting  the  progress  of  digestion.  In  which  tube  do 
we  find  the  most  satisfactory  digestion,  and  why?  How  do  the  indi- 
cations of  the  digestion  of  fibrin  by  trypsin  dift'er  from  the  indications 
of  the  digestion  of  fibrin  by  pepsin  ? 

2.  The  Most  Favorable  Temperature. — (For  this  and  the  fol- 
lowing series  of  experiments  under  tryptic  digestion  use  the  neutral 
extract  plus  an  equal  volume  of  0.5  per  cent  sodium  carbonate.)  In 
each  of  four  tubes  place  5  c.c.  of  alkaline  pancreatic  extract.  Immerse 
one  tube  in  cold  water  from  the  faucet,  keep  a  second  at  room  tempera- 
ture and  place  a  third  on  the  water-bath  at  40°  C.  Boil  the  contents 
of  the  fourth  for  a  few  moments,  then  cool  and  also  keep  it  at  40°  C. 
Into  each  tube  introduce  a  small  piece  of  fibrin  and  note  the  progress 
of  digestion.  In  which  tube  does  the  most  rapid  digestion  occur? 
What  is  the  reason  ? 

3.  Influence  of  Metallic  Salts,  Etc. — Prepare  a  series  of  tubes  and 
into  each  tube  place  6  volumes  of  water,  3  volumes  of  alkaline  pancre- 
atic extract  and  i  volume  of  one  of  the  chemicals  listed  in  Experiment 
18  under  Salivary  Digestion,  page  58. 

Introduce  a  small  piece  of  fibrin  into  each  of  the  tubes  and  keep 
them  at  40°  C.  for  one-half  hour.  Shake  the  tubes  frequently.  In 
which  tubes  do  we  get  the  least  digestion  ? 

4.  Influence  of  Bile. — ^Prepare  five  tubes  as  follows: 
{a)  Five  c.c.  of  pancreatic  extract -fi/2-1  c.c.  of  bile. 
{b)  Five  c.c.  of  pancreatic  extract +1-2  c.c.  of  bile, 
(c)  Five  c.c.  of  pancreatic  extract -f  2-3  c.c.  of  bile. 
{d)  Five  c.c.  of  pancreatic  extract -f  5  c.c.  of  bile. 

(e)  Five  c.c.  of  pancreatic  extract. 

Introduce  into  each  tube  a  small  piece  of  fibrin  and  keep  them  at 
40°  C.  Shake  the  tubes  frequently  and  note  the  progress  of  digestion. 
Does  the  presence  of  bile  retard  tryptic  digestion?  How  do  these 
results  agree  with  those  obtained  under  gastric  digestion  ? 

Experiments  on  Pancreatic  Amylase. 

I.  The  Most  Favorable  Reaction. — Prepare  seven  tubes  as 
follows : 

(a)  One  c.c.  oi  neutral  pancreatic  extract -fi  c.c.  of  starch  paste  -f- 
2  c.c.  of  water. 

{h)  One  c.c.  of  neutral  pancreatic  extract -fi  c.c.  of  starch  paste  + 
2  c.c.  of  I  per  cent  sodium  carbonate. 


144  PHYSIOLOGICAL    CHEMISTRY. 

(r)  One  c.c.  of  neutral  pancreatic  extract +i  c.c.  of  starch  paste  + 
2  c.c.  of  0.5  per  cent  sodium  carbonate. 

((/)  One  c.c.  of  neutral  pancreatic  extract +  1  c.c.  of  starch  paste  + 
2  c.c.  of  0.2  per  cent  hydrochloric  acid. 

{e)  One  c.c.  of  neutral  pancreatic  extract  +  i  c.c.  of  starch  paste  + 
2  c.c.  of  0.2  per  cent  combined  hydrochloric  acid. 

(/)  One  c.c.  of  neutral  pancreatic  extract +1  c.c.  of  starch  paste  + 
2  c.c.  of  0.4  per  cent  boric  acid. 

{g)  One  ex.  of  neutral  pancreatic  extract +1  c.c.  of  starch  paste + 
2  c.c.  of  0.4  per  cent  acetic  acid. 

Shake  each  tube  thoroughly  and  place  them  on  the  water-bath  at 
40°  C.  At  the  end  of  a  half-hour  divide  the  contents  of  each  tube 
into  two  parts  and  test  one  part  by  the  iodine  test  and  the  other  part  by 
Fehling's  test.  Where  do  you  find  the  most  satisfactory  digestion? 
How  do  the  results  compare  with  those  obtained  from  the  similar 
series  under  Trypsin,  page  142  ? 

2.  The  Most  Favorable  Temperature. — (For  this  and  the  fol- 
lowing series  of  experiments  upon  pancreatic  amylase  use  the  neutral 
extract  plus  an  equal  volume  of  0.5  per  cent  sodium  carbonate.)  In 
each  of  four  tubes  place  2-3  c.c.  of  alkaline  pancreatic  extract.  Immerse 
one  tube  in  cold  water  from  the  faucet,  keep  a  second  at  room  tempera- 
ture, and  place  a  third  on  the  water-bath  at  40°  C.  Boil  the  contents 
of  the  fourth  for  a  few  moments,  then  cool  and  also  keep  it  at  40°  C. 
Into  each  tube  introduce  2-3  c.c.  of  starch  paste  and  note  the  progress 
of  digestion.  At  the  end  of  one-half  hour  divide  the  contents  of  each 
tube  into  two  parts  and  test  one  part  by  the  iodine  test  and  the  other 
part  by  Fehling's  test.  In  which  tube  do  you  find  the  most  satisfactory 
digestion  ?  How  does  this  result  compare  with  the  result  obtained 
in  the  similar  series  of  exjjerimcnts  under  Tryjjsin  (see  ])age  143)  ? 

3.  Influence  of  Metallic  Salts,  etc.  Preiiare  a  series  of  tubes 
and  into  each  place  3  volumes  of  water,  3  \'()lumes  of  alkaline  pancreatic 
extract,  i  volume  of  one  of  the  chemicals  listed  in  Experiment  t8  under 
Salivary  Digestion,  page  58,  and  3  volumes  of  starch  paste.  Be  sure 
to  introduce  the  starch  paste  into  the  tube  last.  Why?  Shake  the 
tubes  well  and  fjJace  them  on  the  water-bath  at  40°  C.  At  the  end  of 
a  half-hour  divirle  the  c(jnlents  of  each  tube  into  two  parts  and  test 
(ine  part  by  the  iodine  test  and  the  other  part  by  Fehling's  test.  What 
are  your  conclusions? 

4.  Influence  of  Bile.-  Pre])are  five  tubes  as  follows: 

(a)  2-3  c.c.  <jf  ]>an(  reatic  extract  +2-3  c.c.  of  starch  paste  -|  1/2-1 
c.c.  of  bile. 


PANCREATIC    DIGESTION.  r45 

(b)  2—3  c.c.  of  pancreatic  extract  +  2—3  c.c.  of  starch  paste  + 1—2 
c.c.  of  bile. 

(c)  2-3  c.c.  of  pancreatic  extract  +2-3  c.c.  of  starch  paste  +2-7, 
c.c.  of  bile. 

(d)  2-3  c.c.  of  pancreatic  extract  +2-3  c.c.  of  starch  paste  +5  c.c. 
of  bile. 

(e)  2-3  c.c.  of  pancreatic  extract  +  2-3  c.c.  of  starch  paste. 
Shake  the  tubes  thoroughly  and  place  them  on  the  water-bat;h  at 

40°  C.  Note  the  progress  of  digestion  frequently  and  at  the  end  of  a 
half-hour  divide  the  contents  of  each  tube  into  two  parts  and  test  one 
part  by  the  iodine  test  and  the  other  part  by  Fehling's  test.  What  are 
your  conclusions  regarding  the  influence  of  bile  upon  the  action  of 
pancreatic  amylase  ? 

5.  Digestion  of  Dry  Starch. — To  a  little  dry  starch  in  a  test-tube 
add  about  5  c.c.  of  pancreatic  extract  and  place  the  tube  on  the  water- 
bath  at  40°  C.  At  the  end  of  a  half-hour  filter  and  test  separate  por- 
tions of  the  filtrate  by  the  iodine  and  Fehling  tests.  What  do  you  con- 
clude regarding  the  action  of  pancreatic  amylase  upon  dry  starch? 
Compare  this  result  with  that  obtained  in  the  similar  experiment  under 
Salivary  Digestion  (page  57). 

6.  Digestion  of  Inulin. — To  5  c.c.  of  inulin  solution  in  a  test-tube 
add  10  drops  of  pancreatic  extract  and  place  the  tube  on  the  water- 
bath  at  40°  C.  After  one-half  hour  test  the  solution  by  Fehling's  test.^ 
Is  any  reducing  substance  present  ?  What  do  you  conclude  regarding 
the  digestion  of  inulin  by  pancreatic  amylase  ? 

Experiments  on  Pancreatic  Lipase. 

1.  "Litmus -milk"  Test. — Into  each  of  two  test-tubes  introduce 
10  c.c.  of  milk  and  a  small  amount  of  litmus  powder.  To  the  con- 
tents of  one  tube  add  3  c.c.  of  neutral  pancreatic  extract  and  to  the  con- 
tents of  the  other  tube  add  3  c.c.  of  water  or  of  boiled  neutral  pancre- 
atic extract.  Keep  the  tubes  at  40°  C.  and  note  any  changes  which 
may  occur.     What  is  the  result  and  how  do  you  explain  it  ? 

2.  Ethyl  Butyrate  Test. — Into  each  of  two  test-tubes  introduce 
4  c.c.  of  water,  2  c.c.  of  ethyl  butyrate,  CgH^COO.CjH^,  and  a  small 
amount  of  litmus  powder.  To  the  contents  of  one  tube  add  4  c.c.  of 
neutral  pancreatic  extract  and  to  the  contents  of  the  other  tube  add  4 
c.c.  of  water  of  boiled  neutral  pancreatic  extract.     Keep  the  tubes  at 

'  If  the  inulin  solution  gives  a  reduction  before  being  acted  upon  by  the  pancreatic 
juice,  it  will  be  necessary'  to  determine  the  extent  of  the  original  reduction  by  means  of  a 
"check"  test  (see  page  47). 


I^t)  PHYSIOLOGICAL    CHEMISTRY. 

40*^  C.  and  observe  any  changes  which  may  occur.  What  is  the  result 
and  how  do  you  explain  it?  Write  the  equation  for  the  reaction  which 
has  taken  place. 

Experiments  ox  Pancreatic  Rennin. 

Prepare  four  test-tubes  as  follows: 

(a)  Five  c.c.  of  milk  +  10  drops  of  neutral  pancreatic  extract. 

(b)  Five  c.c.  of  milk  +  20  drops  of  neutral  pancreatic  extract. 

(c)  Five  c.c.  of  milk  +  10  drops  of  alkaline  pancreatic  extract, 
(rf)  Five  c.c.  of  milk  +20  drops  of  alkaline  pancreatic  extract. 
Place  the  tubes  at  6o°-65°  C.  for  a  half  hour  without  shaking. 

Note  the  formation  of  a  clot.^  How  does  the  action  of  pancreatic 
rennin  compare  with  the  action  of  the  gastric  rennin  ? 

'  This  reaction  will  not  always  succeed,  owing  to  conditions  which  are  not  well  under- 
stood. 


CHAPTER  IX. 
BILE. 

The  bile  is  secreted  continuously  by  the  liver  and  passes  into  the 
intestine  through  the  common  bile  duct  which  opens  near  the  pylorus. 
Bile  is  not  secreted  continuously  into  the  intestine.  In  a  fasting  animal 
no  bile  enters  the  intestine,  but  when  food  is  taken  the  bile  begins  to 
flow;  the  length  of  time  elapsing  between  the  ingestion  of  the  food 
and  the  secretion  of  the  bile  as  well  as  the  qualitative  and  quantitative 
characteristics  of  the  secretion  depending  upon  the  nature  of  the  food 
ingested.  Fats,  the  extractives  of  meat  and  the  protein  end-products 
of  gastric  digestion  (proteoses  and  peptones),  cause  a  copious  secretion 
of  bile,  whereas  such  substances  as  water,  acids  and  boiled  starch  paste 
fail  to  do  so.  In  general  a  rich  protein  diet  is  supposed  to  increase  the 
amount  of  bile  secreted,  whereas  a  carbohydrate  diet  would  cause  a 
much  less  decided  increase  and  might  even  tend  to  decrease  the  amount. 
It  has  been  demonstrated  by  Bayliss  and  Starling  that  the  secretion  of 
bile  is  under  the  control  of  the  same  mechanism  that  regulates  the  flow 
of  pancreatic  juice  (see  p.  137).  In  other  words,  the  hydrochloric 
acid  of  the  chyme,  as  it  enters  the  duodenum  transforms  prosecretin 
into  secretin  and  this  in  turn  enters  the  circulation,  is  carried  to  the 
liver,  and  stimulates  the  bile-forming  mechanism  to  increased  activity. 

We  may  look  upon  the  bile  as  an  excretion  as  well  as  a  secretion.  In 
the  fulfillment  of  its  excretory  function  it  passes  such  bodies  as  lecithin, 
metallic  substances,  cholesterol,  and  the  decomposition  products  of 
hcemoglobin  into  the  intestine  and  in  this  way  aids  in  removing  them 
from  the  organism.  The  bile  assists  materially  in  the  absorption  of 
fats  from  the  intestine  by  its  solvent  action  on  the  fatty  acids  formed 
by  the  action  of  the  pancreatic  juice. 

The  bile  is  a  ropy,  viscid  substance  which  is  alkaline  in  reaction 
to  litmus,^  and  ordinarily  possesses  a  decidedly  bitter  taste.  It  varies 
in  color  in  the  different  animals,  the  principal  variations  being  yellow, 
brown,  and  green.  Fresh  human  bile  from  the  living  organism  ordi- 
narily has  a  green  or  golden-yellow  color.  Postmortem  bile  is  variable 
in  color.  It  is  very  difficult  to  determine  accurately  the  amount  of 
normal  bile  secreted  during  any  given  period.     For  an  adult  man  it 

^  It  does  not  contain  ^ny  free  hydroxyl  ions,  however. 

147 


148  PHYSIOLOGICAL    CHEMISTRY. 

has  been  variously  estimated  at  from  500  c.c.  to  iioo  c.c.  for  twenty- 
four  hours.  The  specific  gravity  of  the  bile  varies  between  i.oio  and 
1.040,  and  the  freezing-point  is  about  — 0.56°  C.  As  secreted  by  the 
liver,  the  bile  is  a  clear,  limpid  fluid  which  contains  a  relatively  low 
content  of  solid  matter.  Such  bile  would  have  a  specific  gravity  of 
approximately  i.oio.  After  it  reaches  the  gall-bladder,  however,  it 
becomes  mixed  with  mucous  material  from  the  walls  of  the  gall-bladder, 
and  this  process  coupled  with  the  continuous  absorption  of  water 
from  the  bile  has  a  tendency  to  concentrate  the  secretion.  Therefore 
the  bile  as  we  find  it  in  the  gall-bladder,  ordinarily  possesses  a  higher 
specific  gravity  than  that  of  the  freshly  secreted  fluid.  The  specific 
gravity  under  these  conditions  may  run  as  high  as  1.040. 

The  principal  constituents  of  the  bile  are  the  salts  of  the  bile  acids, 
bile  pigments,  neutral  fats,  lecithin,  phosphatides,  and  cholesterol,  besides 
the  salts  of  iron,  copper,  calcium,  and  magnesium.  Zinc  has  also  fre- 
quently been  found  in  traces. 

The  bile  acids,  which  are  elaborated  exclusi\ely  by  the  hepatic 
cells,  may  be  divided  into  two  groups,  the  glycocholic  acid  group  and 
the  taurocholic  acid  group.  In  human  bile  glycocholic  acid  predomi- 
nates, while  taurocholic  acid  is  the  more  abundant  in  the  bile  of  car- 
nivora.  The  bile  acids  are  conjugate  amino-acids,  the  glycocholic 
acid  yielding  glycocoll, 

CH^NH, 

COOH, 

and  cholic  acid  upon  decomposition,  whereas  taurocholic  acid  gives 
rise  to  taurine, 

CH.,NH2 

I 
CH^SO.OH, 

and  cholic  acid  under  like  conditions.  Glycocholic  acid  contains  some 
nitrogen  but  no  sulphur,  whereas  taurocholic  acid  contains  both  these 
elements.  The  sulj;hur  of  the  taurocholic  acid  is  present  in  the  taurine 
(amino-ethyl-sulphonic-acidj,  of  which  it  is  a  characteristic  constit- 
uent. There  are  several  varieties  of  ch(jh*c  acid  and  therefore  we 
have  several  forms  of  glycocholic  and  taurocholic:  acids,  the  variation 
in  constituti';n  de[)cnding  upon  the  nature  of  llic  cholic  acid  which 
enters  into  the  combination.  The  bile  acids  are  ])resent  in  the  bile 
as  sails  of  one  of  the  alkalis,  generally  sodium.  'J'he  sodium  glyco- 
cholate  and  sodium  taurocholate  may  be  isolated  in  crystalline  form, 


BILE.  149 

either  as  balls  or  rosettes  of  fine  needles  or  in  the  form  of  prisms  having 
ordinarily  four  or  six  sides  (Fig.  40,  below).  The  salts  of  the  bile  acids 
are  dextro-rotatory.  Among  other  properties  these  salts  have  the 
power  of  holding  the  cholesterol  and  lecithin  of  the  bile  in  solution. 

Hammarsten  has  demonstrated  a  third  group  of  bile  acids  in  the 
bile  of  the  shark.  This  same  group  very  probably  occurs  in  certain 
other  animals  also.  These  acids  are  very  rich  in  sulphur  and  resemble 
ethereal  sulphuric  acids  inasmuch  as  upon  treatment  with  boiling 
hydrochloric  acid  they  yield  sulphuric  acid. 


Fig.  40. — Bile  Salts. 

The  bile  pigments  are  important  and  interesting  biliary  constit- 
uents. The  following  have  been  isolated:  bilirubin,  biliverdin,  bili- 
fuscin,  biliprasin,  bilihumin,  bilicyanin,  choleprasin,  and  choletelin.  Of 
these,  bilirubin  and  biliverdin  are  the  most  important  and  predominate 
in  normal  bile.  The  colors  possessed  by  the  various  varieties  of  normal 
bile  are  due  almost  entirely  to  these  two  pigments,  the  biliverdin  being 
the  predominant  pigment  in  greenish  bile  and  the  biHrubin  being 
the  principal  pigment  in  lighter  colored  bile.  The  pigments,  other 
than  the  two  just  mentioned,  have  been  found  almost  exclusively  in 
biliary  calculi  or  in  altered  bile  obtained  at  post-mortem  examinations. 

Bilirubin,  which  is  perhaps  the  most  important  of  the  bile  pigments, 
is  apparently  derived  from  the  blood  pigment,  the  iron  freed  in  the 
process  being  held  in  the  liver.  Bilirubin  has  the  same  percentage 
composition  as  haematoporphyrin,  which  may  be  produced  from  hasmatin. 
Tt  is  a  specific  product  of  the  liver  cells,  but  may  also  be  formed  in  other 
parts  of  the  body.  The  pigment  may  be  isolated  in  the  form  of  a 
reddish-yellow  powder  or  may  be  obtained  in  part,  in  the  form  of 


150  PHYSIOLOGICAL    CHEMISTRY. 

reddish-yellow  rhombic  plates  (Fig.  41,  below)  upon  the  spontaneous 
evaporation  of  its  chloroform  solution.  The  crystalline  form  of  bili- 
rubin is  practically  the  same  as  that  of  haematoidin.  It  is  easily  soluble 
in  chloroform,  somewhat  less  soluble  in  alcohol  and  only  slightly  solu- 
ble in  ether  and  benzene.  Bilirubin  has  the  power  of  combining  with 
certain  metals,  particularly  calcium,  to  form  combinations  which  are 
no  longer  soluble  in  the  solvents  of  the  unaltered  pigment.  Upon 
long  standing  in  contact  with  the  air,  the  reddish-yellow  bilirubin  is 
oxidized  with  the  formation  of  the  green  biliverdin.  Bilirubin  occurs 
in  animal  fluids  as  soluble  bilirubin-alkali. 


^ 


0 

#  f 

Fig.  41. — Bilirubin  (H.*;matoidin).     (Ogderi.) 

Solutions  of  l^liruljin  exhibit  no  absorption-bands.  Jf  an  anv 
moniacal  solution  of  bilirubin-alkali  in  water  is  treated  with  a  solu- 
tion of  zinc  chloride,  however,  it  shows  bands  similar  to  those  of 
bilicyanin  (Absorption  Spectra,  Plate  II),  the  two  bands  between  C 
and  D  being  rather  well  defined. 

Biliverdin  is  jnirlicularly  abundant  in  the  bile  of  herbivora.  It 
is  soluble  in  alcohol  and  glacial  acetic  acid  and  insoluble  in  water, 
chloroform,  and  ether.  Biliverdin  is  formed  from  bilirubin  upon 
oxidation.  It  is  an  amorphous  substance,  and  in  this  differs  from 
bilirubin  which  may  be  at  least  partly  crystallized  under  proper  condi- 
tions, iiiliverdin  may  be  obtained  in  the  form  of  a  green  powder, 
in  common  with  bilirubin,  it  may  be  con\ertc(l  into  liydrobilirubin 
by  nascent  hydrogen. 

'i'he  neutral  solution  of  bilicyanin  or  cholecyanin  is  bluish-green 
or  steel-blue  and  possesses  a  blue  fluorescence,  the  alkaline  solution 
is  green  with  nc;  a[)[)reciable  fluorescence  and  the  strongly  acid  so- 
lution is  violet-blue.  The  alkaline  solution  exhibits  three  absor]) 
tion-banrls,  the  flrst  a  dark,  well-defmed  band  between  C  and  I), 
somewhat  nearer  (";  the  second  a  less  sharply  ficlincd   band  extend 


BILE.  151 

ing  across  D  and  the  third  a  rather  faint  band  between  E  and  F, 
near  E  (Absorption  Spectra,  Plate  II).  The  strongly  acid  solution 
exhibits  two  absorption  bands,  both  lying  between  C  and  E  and 
separated  by  a  narrow  space  near  D.  A  third  band,  exceedingly 
faint,  may  ordinarily  be  seen  between  b  and  F. 

Biliary  calculi,  otherwise  designated  as  biliary  concretions  or  gall 
stones,  are  frequently  formed  in  the  gall-bladder.  These  deposits 
may  be  divided  into  three  classes,  cholesterol  calculi,  pigment  calculi, 
and  calculi  made  up  almost  entirely  of  inorganic  material.  This 
last  class  of  calculus  is  formed  principally  of  the  carbonate  and  phos- 
phate of  calcium  and  is  rarely  found  in  man  although  quite  common 
to  cattle.  The  pigment  calculus  is  also  found  in  cattle,  but  is  more 
common  to  man  than  the  inorganic  calculus.  This  pigment  calculus 
ordinarily  consists  principally  of  bilirubin  in  combination  with  calcium; 
biliverdin  is  sometimes  found  in  small  amount.  The  cholesterol  cal- 
culus is  the  one  found  most  frequently  in  man.  These  may  be  formed 
almost  entirely  of  cholesterol,  in  which  event  the  color  of  the  calculus 
is  very  light,  or  they  may  contain  more  or  less  pigment  and  inorganic 
matter  mixed  with  the  cholesterol,  which  tends  to  give  us  calculi  of 
various  colors. 

For  discussion  of  cholesterol  see  page  246. 

Experiments  on  Bile. 

1.  Reaction. — Test  the  reaction  of  fresh  ox  bile  to  litmus. 

2.  Nucleoprotein. — Acidify  a  small  amount  of  bile  with  dilute 
acetic  acid.     A  precipitate  of  nucleoprotein  forms. 

3.  Inorganic  Constituents.— Test  for  chlorides,  sulphates,  and 
phosphates  (see  page  56). 

4.  Tests  for  Bile  Pigments,  (a)  Gmelin's  Test. — To  about 
5  c.c.  of  concentrated  nitric  acid  in  a  test-tube  add  2-3  c.c.  of  diluted 
bile  carefully  so  that  the  two  fluids  do  not  mk.  At  the  point  of  contact 
note  the  various  colored  rings,  green,  blue,  violet,  red  and  reddish- 
yellow.  Repeat  this  test  with  different  dilutions  of  bile  and  observe 
its  delicacy. 

(b)  Rosenbach's  Modification  of  Gmelin's  Test. — Filter  5  c.c.  of 
diluted  bile  through  a  small  filter  paper.  Introduce  a  drop  of  cancen- 
trated  nitric  acid  into  the  cone  of  the  paper  and  note  the  succession  of 
colors  as  given  in  Gmelin's  test. 

(c)  Nakayama's  Reaction. — To  5  c.c.  of  diluted  bile  in  a  test-tube 
add  an  equal  volume  of  a  10  per  cent  solution  of  barium  chloride, 
centrifugate   the   mixture,   pour  off  the  supernatant    fluid,   and    heat 


1.52  PHYSIOLOGICAL    CHEMISTRY. 

the  precipitate  with  2  c.c.  of  Nakayama's  reagent.^  In  the  presence 
•of  bile  pigments  the  sokition  assumes  a  blue  or  green  color. 

(//)  Hupperfs  Reaction. — Thoroughly  shake  equal  volumes  of 
undiluted  bile  and  milk  of  lime  in  a  test-tube.  The  pigments  unite 
with  the  calcium  and  are  precipitated.  Filter  off  the  precipitate, 
wash  it  v.ith  water,  and  transfer  to  a  small  beaker.  Add  alcohol 
acidified  slightly  with  hydrochloric  acid  and  warm  upon  a  water- 
bath  until  the  solution  becomes  colored  an  emerald  green. 

In  examining  urine  for  bile  pigments,  according  to  Steensma,  this 
procedure  may  give  negative  results  even  in  the  presence  of  the  pig- 
ments, owing  to  the  fact  that  the  acid-alcohol  is  not  a  sufftciently 
strong  oxidizing  agent.  He  therefore  suggests  the  addition  of  a  drop 
of  a  o.  5  per  cent  solution  of  sodium  nitrite  to  the  acid-alcohol  mixture 
before  warming  on  the  water-bath.     Try  this  modification  also. 

ie)  Hammarslen's  Reaction. — To  about  5  c.c.  of  Hammarsten's 
reagent-  in  a  small  evaporating  dish  add  a  few  drops  of  diluted  bile. 
•A' green  color  is  produced.  If  more  of  the  reagent  is  now  added  the 
play  of  colors  as  observed  in  Gmelin's  test  may  be  obtained. 

(/)  Smith's  Test. — To  2-3  c.c.  of  diluted  bile  in  a  test-tube  add 
carefully  about  5  c.c.  of  dilute  tincture  of  iodine  (1:10)  so  that  the 
fluids  do  not  mix.  A  play  of  colors,  green,  blue  and  violet,  is  observed. 
In  making  this  test  upon  the  urine  ordinarily  only  the  green  color  is 
observed. 

ig)  Salkowski-Schippers  Reaction. — To  10  c.c.  of  diluted  bile  in  a 
tiest-tube  add  5  drops  of  a  20  per  cent  solution  of  sodium  carbonate 
and  10  drops  of  a  20  per  cent  solution  of  calcium  chloride.  Filter  off 
the  resultant  precipitate  upon  a  hardened  filter-paper  and  wash  it  with 
water.  Remove  the  precij)itate  to  a  small  porcelain  dish,  add  3  c.c. 
•of  an  acid-alcohol  mixture'  and  a  few  drops  of  a  dilute  solution  of 
•sodium  nitrite  and  heat.  The  prcxlucticjn  of  a  green  color  indicates  the 
presence  of  bile  pigments. 

ih)  Bonanno's  Reaction.'^ — Place  5-10  c.c.  of  diluted  bile  in  a  small 
porcelain  evaporating  dish  and  add  a  few  drops  of  Bonanno's  reagent."^ 
.\n  emerald-green  color  will  dcNcloj). 

'  I'rc|jarcd  hy  combining  ()(>  c.c  of  alcohol  and  i  c.i.  u{  fiiminy,  hydrocliloiic  acidcon- 
taininj/ 4  grams  of  ferrif  chloride  per  liter. 

^  Hammarsten's  reagent  is  made  tjy  mi.xing  i  volume  of  25  jjcr  cent  nitric  acid  and  m; 
volumes  of  25  per  cent  hyrlroc  hloric  acid  and  then  adding  1  volume  of  this  acid  niixlure 
to  4  volumes  of  <)5  per  <  ent  alc<jhol. 

,  ' ^' Made  by  adfling  5  c.c.  of  concentrated  hydrochloric  acid  to  95  c.c.  of  i/j  per  cent 
alv<>h<>l. 
.'•    * //  Tommasi,  2,  N<j.  2r. 

*'ThJs  reagent  may  be  prepared  by  dissolving  2  grams  of  sodium  nitrite  in  100  c.c.  of 
concentrated  iiy<lr<j(  hloric  a(id. 


BILE.  153 

5.  Tests  for  Bile  Acids,  (a)  Pettenkofer's  Test. — To  5  c.c. 
of  diluted  bile  in  a  test-tube  add  5  drops  of  a  5  per  cent  solution  of 
sucrose.  Now  run  about  2-3  c.c.  of  concentrated  sulphuric  acid 
carefully  down  the  side  of  the  tube  and  note  the  red  ring  at  the  point 
of  contact.  Upon  slightly  agitating  the  contents  of  the  tube  the  whole 
solution  gradually  assumes  a  reddish  color.  As  the  tube  becomes 
warm,  it  should  be  cooled  in  running  water  in  order  that  the  tempera- 
ture of  the  solution  may  not  rise  above  70°  C. 

(b)  Mylms's  Modification  of  Pettenkofer's  Test. — To  approxi- 
mately 5  c.c.  of  diluted  bile  in  a  test-tube  add  3  drops  of  a  very  dilute 
(i :  1000)  aqueous  solution  of  furfurol, 

HC  —  CH 

HC        CCHO. 

O 

Now  run  about  2-3  c.c.  of  concentrated  sulphuric  acid  carefully  down 
the  side  of  the  tube  and  note  the  red  ring  as  above.  In  this  case,  also, 
upon  shaking  the  tube  the  whole  solution  is  colored  red.  Keep  the 
temperature  of  the  solution  below  70°  C.  as  before. 

(c)  Neukomm's  Modification  of  Pettenkofer's  Test. — To  a  few 
drops  of  diluted  bile  in  an  evaporating  dish  add  a  trace  of  a  dilute 
sucrose  solution  and  one  or  more  drops  of  dilute  sulphuric  acid. 
Evaporate  on  a  water-bath  and  note  the  development  of  a  violet 
color  at  the  edge  of  the  evaporating  mixture.  Discontinue  the  evapora- 
tion as  soon  as  the  color  is  observed. 

(d)  V.  Udrcinsky's  Test. — To  5  c.c.  of  diluted  bile  in  a  test-tube 
add  3-4  drops  of  a  very  dilute  (1:1000)  aqueous  solution  of  furfurol. 
Place  the  thumb  over  the  top  of  the  tube  and  shake  the  tube  until 
a  thick  foam  is  formed.  By  means  of  a  small  pipette  add  2-3  drops 
of  concentrated  sulphuric  acid  to  the  foam  and  note  the  dark  pink 
coloration  produced. 

(e)  Guerin's  Reaction. — To  equal  volumes  of  diluted  bile  and 
alcohol  in  a  test-tube  add  5-6  drops  of  a  saturated  aqueous  solution 
of  furfurol  and  5-6  drops  of  concentrated  sulphuric  acid.  A  blue 
color  indicates  bile  acids. 

(/)  Hay's  Test. — This  test  is  based  upon  the  principle  that  bile 
acids  have  the  property  of  reducing  the  surface  tension  of  fluids  in 
which  they  are  contained.  The  test  is  performed  as  follows:  Cool 
about  ID  c.c.  of  diluted  bile  in  a  test-tube  to  17°  C.  or  lower  and  sprinkle 


154 


PHYSIOLOGICAL    CHEMISTRY. 


a  little  finely  pulverized  sulphur  upon  the  surface  of  the  fluid.  The 
presence  of  bile  acids  is  indicated  if  the  sulphur  sinks  to  the  bottom 
of  the  liquid,  the  rapidity  with  which  the  sulphur  sinks  depending 
upon  the  quantity  of  bile  acids  present  in  the  mixture.  The  test  is 
said  to  react  with  bile  acids  when  they  arc  present  in  the  proportion 
I  :  120,000. 

Some  investigators  claim  that  it  is  impossible  to  differentiate 
between  bile  acids  and  bile  pigments  by  this  test. 

6.  Crystallization  of  Bile  Salts. — To  25  c.c.  of  undiluted  bile 
in  an  evaporating  dish  add  enough  animal  charcoal  to  form  a  paste 
and  evaporate  to  dryness  on  a  water-bath.  Remove  the  residue, 
grind  it  in  a  mortar,  and  transfer  it  to  a  small  flask.  Add  about  50  c.c. 
of  95  per  cent  alcohol  and  boil  on  a  water-bath  for  20  minutes.  Filter, 
and  add  ether  to  the  filtrate  until  there  is  a  slight  permanent  cloudiness. 
Cover  the  vessel  and  stand  it  away  until  crystallization  is  complete. 
Examine  the  crystals  under  the  microscope  and  compare  them  with 
those  shown  in  Fig.  40,  page  149.  Try  one  of  the  tests  for  bile  acids 
upon  some  of  the  crystals. 

7.  Analysis  of  Biliary  Calculi. — Grind  the  calculus  in  a  mortar 
with  10  c.c.  of  ether.     Filter. 


Filtrate  I. 


.\llo\v  to  evaporate  and  examine  for 
cholesterol   crystals    (Fig.    42,    p.    155). 
(For   further   tests  see    Fxperiment    8, 
Pi55-)      


Residue  I. 

(On  paper  and  in  mortar.) 

Treat   with  diluU-   hydrochloric  acid  and 
filter. 


Filtrate  II. 

Test  for  calcium,  phosphates, 
and  iron.  Evaporate  remainder 
of  filtrate  to  dryness  in  porcelain 
crucible  and  ignite.  Dissolve 
residue  in  dilute  hyrlrochloric 
acid  and  make  alkaline  with 
ammonium  hydroxide.  Blue 
color  indif  ates  copper. 


Residue  II. 

(On  paper  and  in  mortar.) 
Wash  with  a  little  water.     Dry  tiie  filter  paper. 


Treat  with  5  c.c.  chloroform  and  filter. 


Filtrate  III. 


Residue  III. 


Bilirubin.  (On    papi-r  and    in    nior 

(Apply  test  for  bile     tar.)  I 

pigments.)  | 

Treat    with  5   (  .c.   of  hot 
al.ohol. 


liiliverdin. 


BILE. 


155 


8.  Tests  for  Cholesterol. 

(a)  Microscopical  Examination. — Examine  the  crystals  under 
the  microscope  and  compare  them  with  those  shown  in  Fig.  42, 
below. 

{h)  Iodine-sulphuric  Acid  Test. — Place  a  few  crystals  of  cholesterol 
in  one  of  the  depressions  of  a  test-tablet  and  treat  with  a  drop  of 
concentrated  sulphuric  acid  and  a  drop  of  a  very  dilute  solution  of 
iodine.     A  play  of  colors  consisting  of  violet,  blue,  green,  and  red  results. 

{c)  The  Liehermann-Burchard  Test. — Dissolve  a  few  crystals  of 
cholesterol  in  2  c.c.  of  chloroform  in  a  dry  test-tube.  Now  add  10 
drops  of  acetic  anhydride  and  1-3  drops  of  concentrated  sulphuric  acid. 
The  solution  becomes  red,  then  blue,  and  finally  bluish-green  in  color. 


Fig.  42. — Cholesterol. 

{d)  Salkowski's  Test. — Dissolve  a  few  crystals  of  cholesterol  in 
a  little  choloroform  and  add  an  equal  volume  of  concentrated  sulphuric 
acid.  A  play  of  colors  from  bluish-red  to  cherry-red  and  purple  is  noted 
in  the  choloroform  while  the  acid  assumes  a  marked  green  fluorescence. 

{e)  Schiff^s  Reaction. — To  a  little  cholesterol  in  an  evaporating 
dish  add  a  few  drops  of  Schiff's  reagent.^  Evaporate  to  dryness 
over  a  low  flame  and  observe  the  reddish-violet  residue  which  changes 
to  a  bluish-violet. 

9.  Preparation  of  Taurine. — To  300  c.c.  of  bile  in  a  casserole 
add  100  c.c.  of  hydrochloric  acid  and  heat  until  a  sticky  mass  (dyslysin) 
is  formed.  This  point  may  be  determined  by  drawing  out  a  thread- 
like portion  of  the  mass  by  means  of  a  glass  rod,  and  if  it  solidifies 

'  Schiff's  reagent  consists  of  a  mixture  of  three  volumes  of  concentrated  sulphuric  acid 
and  one  volume  of  lo  per  cent  ferric  chloride. 


is6 


PHYSIOLOGICAL   CHEMISTRY. 


immediately  and  assumes  a  brittle  character  we  may  conclude  that  all 
the  taurocholic  and  glycocholic  acid  has  been  decomposed.  Decant 
the  solution  and  concentrate  it  to  a  small  volume  on  the  water-bath. 
Filter  the  hot  solution  to  remove  sodium  chloride  and  other  substances 
which  may  have  separated,  and  evaporate  the  filtrate  to  dryness. 
Dissolve  the  residue  in  5  per  cent  hydrochloric  acid  and  precipitate 
with  ten  volumes  of  95  per  cent  alcohol.  Filter  off  the  taurine  and 
recrystallize  it  from  hot  water.  (Save  the  alcoholic  filtrate  for  the 
preparation  of  glycocolb  below.)  Make  the  following  tests  upon  the 
taurine  crystals. 

(a)  Examine  them  under  the  microscope  and  compare  with  Fig.  43. 


Fig.  43. — Taurine. 


(b)  Heat  a  crystal  upon  platinum  foil.  The  taurine  at  first  melts, 
then  turns  brown,  and  finally  carbonizes  as  the  temperature  is  raised. 
Note  the  suflfocating  odor.     What  is  it  ? 

(c)  Test  the  solubility  of  the  crystals  in  water  and  in  alcohol. 

(d)  Grind  up  a  crystal  with  f(nir  times  its  volume  of  dry  sodium 
carbonate  and  fuse  on  platinum  foil.  Cool  the  residue,  transfer 
it  to  a  test-tube,  and  dissolve  it  in  water.  Add  a  little  dilute  sulphuric 
acid  and  note  the  odor  of  hydrogen  sulphide.  Hold  a  piece  of  filter 
paper,  m(;istcned  with  a  small  amcjunt  of  lead  acetate,  over  the  opening 
of  the  test-tube  and  observe  the  formation  of  lead  sulphide. 

10.  Preparation  of  Glycocoll.  ("(jncentrate  the  alcoholic  filtrate 
from  the  last  experiment  (<))  until  no  more  alcohol  remains.  The 
glycocoll  is  present  here  in  the  fcjrm  of  an  hydrochloride  and  may 
be  liberated  from  this  combination  by  the  addition  of  freshly  ])recipi- 


BILE.  157 

tated  lead  hydroxide  or  by  lead  hydroxide  solution.  Remove  the  lead 
by  hydrogen  sulphide.  Filter  and  decolorize  the  filtrate  by  animal 
charcoal.  Filter  again,  concentrate  the  filtrate,  and  set  it  aside  for 
crystallization.     GlycocoU  separates  as  colorless  crystals  (Fig.  44). 

II.  Synthesis  of  Hippuric  Acid. — To  some  of  the  glycocoU  pre- 
pared in  the  last  experiment  or  furnished  by  the  instructor,  add  a 
little  water,  about  i  c.c.  of  benzoyl  chloride  and  render  alkaline  with 
potassium  hydroxide  solution.  Stopper  the  tube  and  shake  it  until 
no  more  heat  is  evolved.  Now  render  strongly  alkaline  with  potassium 
hydroxide  and  shake  the  mixture  until  no  odor  of  benzoyl  chloride  can 
be   detected.     Cool,   acidify   with   hydrochloric   acid,   add   an   equal 


Fig.  44. — Glycocoi.l. 

volume  of  petroleum  ether,  and  shake  thoroughly  to  remove  the  benzoic 
acid.  (Evaporate  this  solution  and  note  the  crystals  of  benzoic  acid. 
Compare  them  with  those  shown  in  Fig.  94,  page  284.)  Decant  the 
ethereal  solution  into  a  porcelain  dish  and  extract  again  with  ether. 
The  hippuric  acid  remains  in  the  aqueous  solution.  Filter  it  off  and 
wash  it  with  a  small  amount  of  cold  water  while  still  on  the  filter. 
Remove  it  to  a  small,  shallow  vessel,  dissolve  it  in  a  small  amount 
of  hot  water  and  set  it  aside  for  crystallization.  Examine  the  crystals 
microscopically  and  compare  them  with  those  in  Fig.  92,  page  276. 
The  chemistry  of  the  synthesis  is  represented  thus: 

CH,NH3  COCl  OCNHCH,COOH. 

/\  /\ 

+    1=11  +  HCl. 

COOH  \/  \/ 

GlycocoU.  Benzoyl  chloride.        Hippuric  acid. 


CHAPTER  X. 
PUTREFACTION  PRODUCTS. 

The  putrefactive  processes  in  the  intestine  arc  the  result  of  the 
action  of  bacteria  upon  the  protein  material  present.  This  bacterial 
action  which  is  the  combined  effort  of  many  forms  of  micro-organisms 
is  confined  almost  exclusively  to  the  large  intestine.  Some  of  the 
products  of  the  putrefaction  of  proteins  are  identical  with  those  formed 
in  tryptic  digestion,  although  the  decomposition  of  the  protein  material 
is  much  more  extensive  when  subjected  to  putrefaction.  Some  of  the 
more  important  of  the  putrefaction  products  are  the  following:  Indole, 
skatole,  paracresol,  phenol,  para-oxyphenylpropionic  acid,  para-oxyphenyl- 
acetic  acid,  volatile  fatty  acids,  hydrogen  sulphide,  methane,  methyl 
mercaptan,  hydrogen,  and  carbon  dioxide,  beside  proteoses,  peptones, 
ammonia,  and  amino  acids.  Of  these  the  indole,  skatole,  phenol,  and 
paracresol  appear  in  part  in  the  urine  as  ethereal  sulphuric  acids, 
whereas  the  oxyacids  mentioned  pass  unchanged  into  the  urine. 
The  potassium  indoxyl  sulphate  (page  273)  content  of  the  urine  is  a 
rough  indicator  of  the  extent  of  the  putrefaction  within  the  intestine. 

The  portion  of  the  indole  which  is  excreted  in  the  urine  is  first 
subjected  to  a  series  of  changes  within  the  organism  and  is  subsequently 
eliminated  as  indican.     These  changes  may  be  represented  thus: 


NH 

Inrlolu 


('(OH)  /"N  C(OS().,H) 


\/\/CH  \/\/CH 

NH  NH 

Fnfloxyl.  Indoxyl  sulphuric  aciil. 

In  the  presence  of  potassium  salts  the  indoxyl  sulphuric  acid  is 
then  transformed  into  indoxyl  potassium  sulj^hate  (or  indican), 

158 


PUTREFACTION    PRODUCTS.  1 59 

C(0-S03K), 


and  eliminated  as  such  in  the  urine. 

Indican  may  be  decomposed  by  treatment  with  concentrated  hy- 
drochloric acid  (see  tests  on  page  275)  into  sulphuric  acid  and  in- 
doxyl.  The  latter  body  may  then  be  oxidized  to  form  indigo-blue 
thus: 

_C(OH) 

II  +20  = 

/CH 


NH  NH  NH 

Indoxyl.  Indigo-blue. 

This  same  reaction  may  also  occur  under  pathological  conditions 
within  the  organism,  thus  giving  rise  to  the  appearance  of  crystals 
of  indigo-blue  in  the  urine. 

Skatole  is  likewise  changed  within  the  organism  and  eliminated 
in  the  form  of  a  chromogenic  substance.  Skatole  is,  however,  of 
less  importance  as  a  putrefaction  product  than  indole  and  ordinarily 
occurs  in  much  smaller  amount.  The  tryptophane  group  of  the 
protein  molecule  yields  the  indole  and  skatole  formed  in  intestinal 
putrefaction,  but  the  reasons  for  the  transformation  of  the  major 
portion  of  this  tryptophane  into  indole  and  the  minor  portion  into 
skatole  are  not  well  understood.     Indole  is  more  toxic  than  skatole. 

Phenol  occurs  in  fairly  large  amount  in  certain  abnormal  con- 
ditions of  the  organism,  but  ordinarily  the  amount  is  very  small. 
It  is  probably  derived  from  the  tyrosine  group  of  the  protein  mole- 
cule. Phenol  is  conjugated  in  the  liver  to  form  phenyl  potassium 
sulphate  and  appears  in  the  urine  in  this  form  (Baumann  and  Herter). 
Para-cresol  occurs  in  the  urine  as  cresyl  potassium  sulphate. 

Regarding  the  claim  of  Nencki  that  methyl  mercaptan  is  formed 
as  a  gas  during  intestinal  putrefaction  it  is  an  important  fact  that 
Herter^  has  been  unable  to  detect  the  mercaptan  in  fresh  feces.  He 
is,  therefore,  not  inclined  to  accept  the  theory  that  methyl  mercap- 
tan is  formed  in  ordinary  intestinal  putrefaction  but  believes  that  it 
may  be  formed  in  exceptional  cases.  Hydrogen  sulphide  is,  however, 
formed  in  all  cases  of  intestinal  putrefaction. 

'  Herter:     Bacterial  Infections  of  the  Digestive  Tract,  p.  227. 


l6o  PHYSIOLOGICAL    CHEMISTRY. 

Experiments  ox  Putrefaction  Products. 

In  many  courses  in  physiological  chemistry  the  instructors  are  so 
limited  for  time  that  no  extended  study  of  the  products  of  putre- 
faction can  very  well  be  attempted.  Under  such  conditions  the  scheme 
here  submitted  may  be  used  profitably  in  the  way  of  a  demonstration. 
\Vhere  the  number  of  students  is  not  too  great,  a  single  large  putrefac- 
tion may  be  started,  and,  after  the  initial  distillation,  both  the  result- 
ing distillate  and  residue  may  be  distributed  to  the  members  of  the  class 
for  indi\idual  manipulation. 

Preparation  of  Putrefaction  Mixture. — Place  a  weighed  mix- 
ture of  coagulated  egg  albumin  and  ground  lean  meat  in  a  flask  or 
bottle  and  add  approximately  2  liters  of  water  for  every  kilogram 
of  protein  used.  Sterilize  the  vessel  and  contents,  inoculate  with 
the  colon  bacillus,  and  keep  at  40°  C.  for  two  or  three  weeks.  If  cultures 
of  the  colon  bacillus  are  not  available,  add  60  c.c.  of  a  cold  saturated 
solution  of  sodium  carbonate  for  every  liter  of  water  previously  added 
and  inoculate  with  some  putrescent  material  (pancreas  or  feces). ^  Mix 
the  putrefaction  mixture  very  thoroughly  by  shaking  and  insert  a  cork 
furnished  with  a  glass  tube  to  which  is  attached  a  wash  bottle  contain- 
ing a  3  per  cent  solution  of  mercuric  cyanide.^  This  device  is  for  the 
purpose  of  collecting  the  methyl  mercaptan,  a  gas  formed  during  the 
process  of  putrefaction.  It  also  serves  to  diminish  the  odor  arising 
from  the  putrefying  material.  Place  the  putrefaction  mixture  at  40° 
C.  for  two  or  three  weeks  and  at  the  end  of  that  time  make  a  separation 
of  the  products  of  putrefaction  according  to  the  following  directions: 

Subject  the  mixture  to  distillation  until  the  distillate  and  residue 
are  approximately  ecjual  in  \ohimc. 

'  Putrefying  protein  may  be  prcpare(]  by  treating  to  grams  of  finely  ground  lean  meat 
with  100  t.c.  of  water  and  2  c.c.  of  a  saturated  solution  of  sodium  carbonate  and  keeping 
the  mixture  at  40°  C.  for  twenty-four  hours. 

^Concentrated  sulphuric  acid  containing  a  small  aniouiil  of  i.uiliii  may  l)c  used  as  a 
substitute  for  mercuric  cyanifie.  When  this  niodilH  alion  is  cinploycd  il  is  necessary 
to  use  calcium  chloride  tubes  to  exclude  moisture  from  the  isaiin  soluiiuii. 


PUTREFACTION    PRODUCTS. 


i6i 


PART  I. 
MANIPULATION  OF  THE  DISTILLATE. 

Acidify  with  hydrochloric  acid  and  extract  with  ether. 


Ether  Extract  Pfo.  i. 

Add  an  equal  volume  of  water,  make 
alkaline  with  potassium  hydroxide,  and 
shake  thoroughly.      I 


Ether  Extract  No.  2. 

Evaporate  spontaneously.  Indole 
and  skatole  remain.  Try  proper  reac- 
tions (see  pages  164  and  166). 


Ether  Extract  No.  3. 

Evaporate.     Detect  phenol  and  cresol 
(paracresol) .     See  p.  166. 


Residue  No.  1 

AUov,-  the  ether  to  volatilize.  Evapo- 
rate and  detect  ammonium  chloride 
ciystals  (Fig.  45,  p.  162). 


Alkaline  Solution  No.  i. 

Acidify  with  hydrochloric  acid,  add 
sodium  carbonate,  and  extract  nith 
ether. 


Alkaline  Solution  No.  2. 

Acidify    with    hydrochloric    acid,    and 
extract  with  ether. 


Ether  Extract  No.  4. 

Evaporate.     Volatile  fatty  acids  re- 


Final  Residue. 

(Discard.) 


DETAILED  DIRECTIONS  FOR  MAKING  THE 

SEPARATIONS  INDICATED  IN 

THE  SCHEME. 

Preliminary  Ether  Extraction. — This  extraction  may  be  con\'eni- 
ently  conducted  in  a  separately  funnel.  Mix  the  fluids  for  extraction 
in  the  ratio  of  two  volumes  of  ether  to  three  volumes  of  the  distillate. 
Shake  very  thoroughly  for  a  few  moments,  then  draw  off  the  extracted 
fluid  and  add  a  new  portion  of  the  distillate.  Repeat  the  process  until 
the  entire  distillate  has  been  extracted.  Add  a  small  amount  of  fresh 
ether  at  each  extraction  to  replace  that  dissolved  by  the  water  in  the 
preceding  extraction. 

Residue  No.  1. — Unite  the  portions  of  the  distillate  extracted  as 
above  and  allow  the  ether  to  volatilize  spontaneously.  Evaporate 
until  crystallization  begins.  Examine  the  crystals  under  the  micro- 
scope.    Ammonium    chloride    predominates.     Explain    its  presence. 


1 62 


PHYSIOLOGICAL   CHEMISTRY. 


Ether  Extract  Xo.  i. — Add  an  equal  volume  of  water,  render  the 
mixture  alkaline  with  potassium  hydroxide,  and  shake  thoroughly  by 
means  of  a  separatory  funnel  as  before.  The  volatile  fatty  acids, 
contained  among  the  putrefaction  products,  would  be  dissolved  by 
the  alkaline  solution  (Xo.  i)  whereas  any  indole  or  skatole  would 
remain  in  the  ethereal  solution  (  No.  2). 


Fig.  45. — Aaimoniuii  Chloride. 


Alkaline  Soluti&n  No.  i. — Acidify  with  hydrochloric  acid  and  add 
sodium  carbonate  solution  until  the  fluid  is  neutral  or  slightly  acid 
from  the  presence  of  carbonic  acid.  At  this  point  a  portion  of  the 
solution,  after  being  heated  for  a  few  moments,  should  possess  an 
alkaline  reaction  on  cooling.  Extract  the  whole  mixture  with  ether 
in  the  usual  way,  using  care  in  the  manipulatic^n  of  the  stop  cock  to 
relieve  the  pressure  due  to  the  evolution  of  carbon  dioxide.  The 
ether  (Ether  Extract  No.  3)  removes  any  phenol  or  cresol  which  may 
be  present  while  the  volatile  fatty  acids  will  remain  in  Ihc  alkaline 
solution  (No.  2)  as  alkali  salts. 

Ether  Extract  No.  2. — Drive  off  the  major  portion  of  the  ether 
at  a  low  temperature  on  a  water-bath  and  allow  the  residue  to  evap- 
orate sjjonluncously.  Indole  and  skatole  should  be  ])resent  here. 
Prove  the  presence  of  these  bodies.  P'or  tests  lor  indole  and  skatole 
see  pp.  164  and  166. 

Alkaline  Solution  No.  2. — Make  strongly  acid  with  hydrochloric 
acid  and  extract  with  a  small  amount  of  ether,  using  a  separatory 
funnel.  As  carbon  dioxide  is  liberated  here,  care  must  be  used  in 
the  manipulation  of  the  stop  cock  of  the  funnel  in  relieving  the  pressure 


PUTREFACTION   PRODUCTS. 


163 


within  the  vessel.  The  volatile  fatty  acids  are  dissolved  by  the  ether- 
(Ether  Extract  No.  4). 

Ether  Extract  No.  3. — Evaporate  this  ethereal  solution  on  a  water- 
bath.  The  oily  residue  contains  phenol  and  cresol.  The  cresol  is 
present  for  the  most  part  as  paracresol.  Add  some  water  to  the  oily 
residue  and  heat  it  in  a  flask.  Cool  and  prove  the  presence  of  phenol 
and  cresol.    For  tests  for  these  bodies  see  page  166. 

Ether  Extract  No.  4. — Evaporate  on  a  water-bath.  The  volatile 
fatty  acids  remain  in  the  residue. 


PART   II. 
MANIPULATION  OF  THE  RESIDUE. 

Evaporate,  filter,  and  extract  with  ether. 


Ether  Extract. 

Evaporate,  extract  the  residue  with 
warm  water,  and  filter. 


Aqueous  Solution. 

Evaporate  until  crystals  begin  to 
form.  Stand  in  a  cold  place  until 
crystallization  is  complete.     Filter. 


Crystalline  Deposit. 

Consists  of  a  mixture  of 
leucine  and  tyrosine  crystals. 
(Figs,  23,  26  and  104,  pages 
7i>  75  and  344-) 


Filtrate  No.  2. 

Contains  oxyacids  and 
skatole-carhonic  acid. 


Residue. 

Contains  non-volatile 
fatty  acids. 


Filtrate  No.  i. 

Contains   proteose,    peptone, 
aromatic  acids,  and  tryptophane . 


DETAILED   DIRECTIONS   FOR  MAKING  THE 

SEPARATIONS   INDICATED   IN 

THE   SCHEME. 

Preliminary  Ether  Extraction. — This  extraction  may  be  conducted 
in  a  separatory  funnel.  In  order  to  make  a  satisfactory  extraction 
the  mixture  should  be  shaken  very  thoroughly.  Separate  the  ethereal 
solution  from  the  aqueous  portion  and  treat  them  according  to  the 
directions  given  on  p.  161. 


164  PHYSIOLOGICAL    CHEMISTRY. 

'Ether  Extract. — Evaporate  this  solution  on  a  safety  water-bath 
until  the  ether  has  been  entirely  removed.  Extract  the  residue  with 
warm  water  and  filter. 

Aqueous  Solution. — Evaporate  this  solution  until  cl"ystallization 
begins.  Stand  the  solution  in  a  cold  place  until  no  more  crystals 
form.  This  crvstalline  mass  consists  of  impure  leucine  and  tyrosine. 
Filter  off  the  crystals. 

Crystalline  Deposit.— Y.xa.mmQ  the  crystals  under  the  microscope 
and  compare  them  with  those  reproduced  in  Figs.  23,  26,  and  104, 
pages  71,  75  and  344.  Do  the  forms  of  the  crystals  of  leucine  and 
tyrosine  resemble  those  previously  examined?  Make  a  separation 
of  the  leucine  and  tyrosine  and  apply  typical  tests  according  to  direc- 
tions given  on  pages  81  and  82. 

Filtrate  No.  i. — Make  a  test  for  tryptophane  with  bromine  water 
(see  page  142),  and  also  with  the  Hopkins-Cole  reagent  (see  page 
89).  Use  the  remainder  of  the  filtrate  for  the  separation  of  proteoses 
and  peptones.  Make  the  separation  according  to  the  directions  given 
on  page  112. 

Filtrate  A^o.  2. — This  solution  contains  para-oxyphenylacetic  acid, 
para-oxyphenylpropionic  acid  and  skatole-carbonic  acid.  Prove  the 
presence  of  these  bodies  by  appropriate  tests.  Tests  for  oxyacids 
and  skatole-carbonic  acid  are  given  on  page  167. 

TESTS  FOR  VARIOUS  PUTREFACTION  PRODUCTS. 
Tests  for  Indole. 

I.  Herter's     ,3'-Naphthaquinone    Reaction. — (a)   To    a    dilute 

aqueous  solution  of  indole  (1:500,000)  add  one  drop  of  a  2  per  cent 
solution  of  /5-naphthaquinone-sodium-monosulphonatc.  No  reaction 
occurs.  Add  a  drop  of  a  ro  ])er  cent  solution  of  ])otassium  hydroxide 
and  note  the  gradual  development  of  a  blue  or  blue-green  color  which 
fades  to  green  if  an  excess  of  the  alkali  is  added.  Render  the  green 
or  blue-green  solution  acid  and  note  the  appearance  of  a  pink  color. 
Heat  facilitates  the  development  of  the  color  reaction. 

One  part  of  indole  in  ofje  million  parts  of  water  may  be  detected 
by  means  of  this  test  if  carefully  performed. 

(b)  U  the  alkali  be  added  to  a  more  concentrated  indole  solution 
before  the  introduction  of  the  naphtha(|uinone  the  course  of  the  re- 
action is  different,  particularly  if  the  indole  solution  is  somewhat 
more  concentrated  than  that  mentioned  above  anrl  if  heal  is  used. 


PUTREFACTION    PRODUCTS.  165 

Under  these  conditions  the  blue  indole  compound  ultimately  forms 
as  fine  acicular  crystals  which  rise  to  the  surface. 

If  we  do  not  wait  for  the  production  of  the  crystalline  body  but  as 
soon  as  the  blue  color  forms,  shake  the  aqueous  solution  with  chloro- 
form, the  blue  color  disappears  from  the  solution  and  the  chloroform 
assumes  a  pinkish-red  hue.  This  is  a  distinguishing  feature  of  the 
indole  reaction  and  facilitates  the  differentiation  of  indole  from  other 
bodies  which  yield  a  similar  blue  color. 

2.  Konto's  Reaction. — Distil  the  solution  to  be  tested  until  only 
one-third  of  the  original  solution  remains.  Make  the  distillate  alkaline 
with  sodium  hydroxide  and  distil  again  in  order  to  separate  the  indole 
from  the  phenol,  the  latter  remaining  in  the  residue.  Inasmuch  as 
this  second  distillate  generally  contains  a  large  amount  of  ammonia 
it  should  be  acidified  with  dilute  sulphuric  acid  and  again  distilled. 
To  I  c.c.  of  this  ammonia-free  distillate  in  a  test-tube  add  3  drops  of 
a  40  per  cent  solution  of  formaldehyde  and  i  c.c.  of  concentrated 
sulphuric  acid.  Now  agitate  the  mixture  and  note  the  appearance 
of  a  violet  red  color  if  a  trace  of  indole  is  present.  The  test  is  said  to 
serve  for  the  detection  of  indole  when  present  in  a  dilution  of  i :  700,000. 

Skatole  gives  a  yellow  or  brown  color  under  the  above  conditions. 

3.  Cholera-red  Reaction.- — To  a  little  of  the  residue  in  a  test- 
tube  add  one-tenth  its  volume  of  a  0.02  per  cent  solution  of  potassium 
nitrite  and  mix  thoroughly.  Carefully  run  concentrated  sulphuric 
acid  down  the  side  of  the  tube  so  that  it  forms  a  layer  at  the  bottom. 
Note  the  purple  color.  Neutralize  with  potassium  hydroxide  and 
observe  the  production  of  a  bluish-green  color. 

4.  Legal's  Reaction. — To  a  small  amount  of  the  residue  in  a 
test-tube  add  a  few  drops  of  a  freshly  prepared  solution  of  sodium 
nitroprusside,  Na3Fe(CN)5NO  +  2H20.  Render  alkaline  with  potas- 
sium hydroxide  and  note  the  production  of  a  violet  color.  If  the 
solution  is  now  acidified  with  glacial  acetic  acid  the  violet  is  trans- 
formed into  a  blue. 

5.  Pine  "Wood  Test. — Moisten  a  pine  splinter  with  concentrated 
hydrochloric  acid  and  insert  it  into  the  residue.  The  wood  assumes 
a  cherry-red  color. 

6.  Nitroso-indole  Nitrate  Test. — Acidify  some  of  the  residue 
with  nitric  acid,  add  a  few  drops  of  a  potassium  nitrite  solution  and 
note  the  production  of  a  red  precipitate  of  nitroso-indole  nitrate.  If 
the  residue  contains  but  little  indole  simply  a  red  coloration  will 
result.  Compare  this  result  with  the  result  of  the  similar  test  on 
skatole. 


1 66  PHYSIOLOGICAL   CHEMISTRY. 

Tests  for  Skatole. 

1.  Herter's    Para-dimethylaminobenzaldehyde    Reaction.*  — 

To  5  c.c.  of  the  distillate  or  aqueous  solution  under  examination 
add  I  c.c.  of  an  acid  solution  of  para-dimethylaminobenzaldehyde^ 
and  heat  the  mixture  to  boiling.  A  purplish-blue  coloration  is  pro- 
duced^ which  may  be  intensified  through  the  addition  of  a  few  drops 
of  concentrated  hydrochloric  acid.  If  the  solution  be  cooled  under 
running  water  it  loses  its  purplish  tinge  of  color  and  becomes  a  definite 
blue.  The  solution  at  this  point  may  be  somewhat  opalescent  through 
the  separation  of  uncombined  para-dimethylaminobenzaldehyde. 
Care  should  be  taken  not  to  add  an  excess  of  hydrochloric  acid  inas- 
much as  the  end-reaction  has  a  tendency  to  fade  under  the  influence 
of  a  high  acidity. 

A  rough  idea  regarding  the  actual  quantity  of  skatole  in  a  mixture 
may  be  obtained  by  extracting  this  blue  solution  with  chloroform 
and  subsequently  comparing  this  chloroform  solution,  l^y  means  of 
a  colorimeter  (Duboscq),  with  the  maximal  reaction,  obtained  with 
a  skatole  solution  of  known  strength. 

2.  Color  Reaction  with  Hydrochloric  Acid. — Acidify  some  of 
the  residue  with  concentrated  hydrochloric  acid.  Note  the  production 
of  a  violet  color. 

3.  Acidify  some  of  the  residue  with  nitric  acid  and  add  a  few  drops 
of  a  potassium  nitrite  solution.  Note  the  white  turbidity.  Compare 
this  result  with  the  result  of  the  similar  test  on  indole. 

Tests  for  Phenol  and  Cresol. 

1.  Color  Test. — Test  a  little  of  the  solution  with  Millon's  reagent. 
A  red  color  results.  Compare  this  test  with  the  similar  one  under 
Tyrosine  (see  page  81). 

2.  Ferric  Chloride  Test. — Add  a  few  drops  of  neutral  ferric 
chlorifle  solution  to  a  little  of  the  residual  fluid.  A  dirty  bluish-gray 
color  is  formed. 

3.  Formation  of  Bromine  Compounds.  ;\dd  some  bromine 
water  to  a  little  of  the  fluid  under  examination.  Note  the  crystalline 
precipitate  of  tribromyjhenol  and  tribromcresol. 

'  Hertcr:     Bacterial  Infedions  of  the  Digestive  Tract,  1907,  p.  141. 

*  Marie  by  dissf>lving  5  grams  of  para-diincthylaminobenzaUlchy<ie  in  100  c.c.  of  10 
per  cent  sulphuric  acid. 

^  If  the  color  docs  not  appear  add  more  of  the  aldehyde  solution. 


PUTREFACTION    PRODUCTS.  1 67 

Tests  for  Oxyacids. 

1.  Color  Test. — Test  a  little  of  the  solution  with  Millon's  reagent. 
A  red  color  results. 

2.  Bromine  Water  Test. — Add  a  few  drops  of  bromine  water 
to  some  of  the  filtrate.    A  turbidity  or  precipitate  is  observed. 

Test  for  Skatole-carbonic  Acid. 

Ferric  Chloride  Test. — Acidify  some  of  the  filtrate  with  hydro- 
chloric acid,  add  a  few  drops  of  ferric  chloride  solution,  and  heat. 
Compare  the  end-reaction  with  that  given  by  phenol. 


CH.\PTER  XL 

FECES. 

The  feces  is  the  residual  mass  of  material  remaining  in  the  intes- 
tine after  the  full  and  complete  exercise  of  the  digestive  and  absorptive 
functions  and  is  ultimately  expelled  from  the  body  through  the  rectum. 
The  amount  of  this  fecal  discharge  varies  with  the  individual  and  the 
diet.  Upon  an  ordinary  mixed  diet  the  daily  excretion  by  an  adult 
male  will  aggregate  1 10-170  grams  with  a  solid  content  ranging 
between  25  and  45  grams;  the  fecal  discharge  of  such  an  individual 


Fig.  46. — MicKuscoi'icAL  Constituexis  of  Feces,     (v.  Jaksch.) 
a.  Muscle  fibers;  b,  connective  tissue;  c,  epithelium;  d,  leucocytes;  e,  spiral  cells;/,  g,  h,  i, 
various  vegetable  cells;  k,  "triple  phosphate"  crystals;  /,  woody  vegetable  cells;  the  whole 
interspersed  with  innumerable  micro-organisms  of  various  kinds. 

upon  a  vegetable  diet  will  be  much  greater  and  may  c'\en  be  as  great 
as  350  grams  and  possess  a  solid  content  (^f  75  grams.  'J'hc  variation 
in  the  normal  daily  output  being  so  great  renders  this  factor  of  very 
little  value  for  diagnostic  purposes,  except  where  the  composition  of 
the  diet  is  accurately  known.  Lesions  of  the  digestive  tract,  a  defective 
absorptive  function,  or  increased  peristalsis  as  well  as  an  admixture 
of  mucus,  pus,  blood,  and  pathological  products  of  the  intestinal  wall 
may  cause  the  total  amount  of  excrement  to  be  markedly  increased. 

'i'he  fecal  pigment  of  the  normal  adult  is  hy(]r()l)ilirul)in.     This 
pigment  originates  from  the  bilirubin  which  is  secreted  into  the  intes- 

168 


FECES.  169 

tine  in  the  bile,  the  transformation  from  bilirubin  to  hydrobilirubin 
being  brought  about  through  the  activity  of  certain  bacteria.  Hydro- 
bilirubin is  sometimes  called  stercobilin  and  bears  a  close  resemblance 
to  urobilin  or  may  even  be  identical  with  that  pigment.  Neither 
bilirubin  nor  biliverdin  occurs  normally  in  the  fecal  discharge  of  adults, 
although  the  former  may  be  detected  in  the  excrement  of  nursing 
infants.  The  most  important  factor,  however,  in  determining  the 
color  of  the  fecal  discharge  is  the  diet.  A  mixed  diet,  for  instance, 
produces  stools  which  vary  in  color  from  light  to  dark  brown,  an 
exclusive  meat  diet  gives  rise  to  a 
brownish-black  stool,  whereas  the 
stool  resulting  from  a  milk  diet  is 
invariably  light  colored.  Certain 
pigmented  foods  such  as  the  chlor- 
ophyllic  vegetables,  and  various 
varieties  of  berries,  each  afford 
stools  having  a  characteristic  color. 

Certain  drugs  act  in  a  similar  wav 

,       xi       r       1   J-     1  rm  •    •'    Fig.  47. — HiiLATOiDiN  Cryst.axs  from 

to  color  the  fecal  discharge.    This  is         acholic  Stools,    {v.  Jaksch.) 

well  illustrated  by  the  occurrence  of  ^°l°^  °f  cn-stals  same  as  the  color  of 

those  in  Fig.  41,  p.  150. 

green    stools    following    the    use    of 

calomel  and  of  black  stools  after  bismuth  ingestion.  The  green 
color  of  the  calomel  stool  is  generally  believed  to  be  due  to  biliverdin. 
V.  Jaksch,  however,  claims  to  have  proven  this  \iew  to  be  incorrect 
since  he  was  able  to  detect  hydrobilirubin  (or  urobilin)  but  no  bili- 
verdin in  stools  after  the  administration  of  calomel.  The  bismuth 
stool  derives  its  color  from  the  black  sulphide  which  is  formed  from 
the  subnitrate  of  bismuth.  In  cases  of  biliary  obstruction  the  gravish- 
white  acholic  stool  is  formed. 

Under  normal  conditions  the  odor  of  feces  is  due  to  skatole  and 
indole,  two  bodies  formed  in  the  course  of  putrefactive  processes 
occurring  within  the  intestine  (see  page  158).  Such  bodies  as  methane, 
methyl  mercaptan,  and  hydrogen  sulphide  may  also  add  to  the  dis- 
agreeable character  of  the  odor.  The  intensity  of  the  odor  depends 
to  a  large  degree  upon  the  character  of  the  diet,  being  verv  marked 
in  stools  from  a  meat  diet,  much  less  marked  in  stools  from  a  vege- 
table diet,  and  frequently  hardly  detectable  in  stools  from  a  milk  diet. 
Thus  the  stool  of  the  infant  is  ordinarily  nearly  odorless  and  anv 
decided  odor  may  generally  be  readily  traced  to  some  pathological 
source. 

A    neutral    reaction    ordinarily    predominates    in    normal    stools 


lyo  PHYSIOLOGICAL    CHEMISTRY. 

although  slightly  alkaline  or  even  acid  stools  are  met  with.  The 
acid  reaction  is  encountered  much  less  frequently  than  the  alkaline 
and  then  commonly  only  following  a  vegetable  diet. 

The  form  and  consistency  of  the  stool  is  dependent,  in  large  measure, 
upon  the  nature  of  the  diet  and  particularly  upon  the  quantity  of 
water  ingested.  Under  normal  conditions  the  consistency  may  vary 
from  a  thin,  pasty  discharge  to  a  firmly  formed  stool.  Stools  which 
are  exceedingly  thin  and  watery  ordinarily  ha\e  a 
pathological  significance.  In  general  the  feces  of 
the  carnivorous  animals  is  of  a  firmer  consistency 
than  that  of  the  herbivora. 

It   is   frequently  desirable  for  clinical  or  experi- 
mental   purposes    to    make    an   examination  of  the 

Fig.  48.— Ch-arcot-  fecal  output  which  constitutes  the  residual  mass  from 
Leyden  Crystals.  .        ,    ^    .         ,.  tt     1  1  i-  • 

a   certam   definite   diet.      Under  such  conditions,  it 

is  customary  to  cause  the  person  under  observation  to  ingest  some 
substance,  at  the  beginning  and  end  of  the  period  in  question,  which 
shall  sufficiently  differ  in  color  and  consistency  from  the  surrounding 
feces  as  to  render  comparatively  easy  the  differentiation  of  the  feces 
of  that  period  from  the  feces  of  the  immediately  preceding  and  suc- 
ceeding periods.  One  of  the  most  satisfactory  methods  of  making 
this  ''separation"  is  by  means  of  the  ingestion  of  a  gelatin  capsule 
containing  about  0.2  gram  of  powdered  charcoal  at  the  beginning  and 
end  of  the  period  under  observation.  This  procedure  causes  the 
appearance  of  two  black  zones  of  charcoal  in  the  fecal  mass  and  thus 
renders  comparatively  simple  the  differentiation  of  the  feces  of  the 
intermediate  period.  Some  similar  method  for  the  "separation  of 
feces"  is  universally  j^ractised  in  connection  with  the  scientifically 
accurate  type  of  nutrition  or  metabolism  experiment  which  embraces 
the  collection  of  useful  data  regarding  the  income  and  outgo  of 
nitrogen,  and  other  elements. 

Among  the  macroscopical  constituents  of  the  feces  may  be  men- 
tioned the  following:  Intestinal  parasites,  undigested  food  particles, 
gall  stones,  pathological  products  of  the  intestinal  wall,  enteroliths, 
intestinal  sand,  and  objects  which  have  been  accidentally  swallowed. 

The  fecal  constituents  which  at  \'arious  times  and  under  different 
conditions  may  be  detected  by  the  use  of  the  microscope  are  as  follows: 
Constituents  derived  from  the  ffjod,  such  as  muscle  fibers,  connective- 
tissue  shreds,  starch  i^ramdcs,  and  fat;  f(jrmed  elements  derived  from 
the  intestinal  tract,  such  as  epithelium,  erythrocytes,  and  leucocytes; 
miicus;  pus  corpuscles;  parasites  and  bacteria,     in  aildilion  to  the  con- 


FECES.  171 

stituents  named  the  following  crystalline  deposits  may  be  detected: 
cholesterol,  soaps,  fatty  acid,  fat,  bismuth  sulphide,  hcematoidin,  "  triple 
phosphate,^''  Char  cot-Ley  den  crystals,  axv^  the  oxalate,  carbonate,  phos- 
phate, sulphate,  and  lactate  of  calcium. 

The  detection  of  minute  quantities  of  blood  in  the  feces  ("occult 
blood")  has  recently  become  a  recognized  aid  to  a  correct  diagnosis 
of  certain  disorders.  In  these  instances  the  hemorrhage  is  ordinarily 
so  slight  that  the  identification  by  means  of  macroscopical  character- 
istics as  well  as  the  microscopical  identification  through  the  detec- 
tion of  erythrocytes  are  both  unsatisfactory  in  their  results.  Of  the 
tests  given  for  the  detection  of  "occult  blood"  the  aloin-tur pen  tine 
test  (page  173)  is  probably  the  most  satisfactory.  Since  "occult  blood" 
occurs  with  considerable  regularity  and  frequency  in  gastrointestinal 
cancer  and  in  gastric  and  duodenal  ulcer,  its  detection  in  the  feces  is 
of  especial  value  as  an  aid  to  a  correct  diagnosis  of  these  disorders. 

It  has  been  quite  clearly  shown  that  the  intestine  of  the  newly 
born  is  sterile.  However,  this  condition  is  quickly  altered  and  bac- 
teria may  be  present  in  the  feces  before  or  after  the  first  ingestion  of 
food.  There  are  three  possible  means  of  infecting  the  intestine,  i.  e., 
by  way  of  the  mouth  or  anus  or  through  the  blood.  The  infection  by 
means  of  the  blood  seldom  occurs  except  under  pathological  condi- 
tions, thus  limiting  the  general  infection  to  the  mouth  and  anus. 

In  infants  with  pronounced  constipation  two-thirds  of  the  dry 
substance  of  the  stools  has  been  found  to  consist  of  bacteria.  In  the 
stools  of  normal  adults  probably  about  one-third  of  the  dry  substance 
is  bacteria.^  The  average  excretion  of  dry  bacteria  in  twenty-four 
hours  for  an  adult  is  about  8  grams. 

Some  of  the  more  important  organisms  met  with  in  the  feces  are 
the  following:^  B.  coli,  B.  lactis  aero  genes,  Bad.  Welchii,  B.  bifidus, 
and  coccal  forms.  Of  these  the  first  three  types  mentioned  are  gas- 
forming  organisms.  The  production  of  gas  by  the  fecal  flora  in  dex- 
trose-bouillon is  subject  to  great  variations  under  pathological  con- 
ditions: alterations  in  the  diet  of  normal  persons  will  also  cause  wide 
fluctuations.  In  this  connection  Herter  has  observed  a  marked 
reduction  or  even  complete  cessation  of  gas  production  by  the  mixed 
fecal  bacteria  while  considerable  doses  of  benzoate  were  being  given. 
A  return  to  the-  former  plane  of  gas  production  followed  the  discon- 
tinuation of  the  benzoate.*     Data  as  to  the  production  of  gas  are  of 

^  Schittenhelm  and  ToUens  found  bacteria  to  comprise  42  per  cent  of  the  drj-  matter. 
This  vahie  is,  however,  probably  too  high. 

^Herter  and  Kendall:     Journal  of  Biological  Chemistry,  190S,  V,  p.  283. 
^  Private  communication  from  Professor  C.  A.  Herter. 


172 


PHYSIOLOGICAL    CHEMISTRY. 


considerable  importance  in  a  diagnostic  way  although  the  exact  cause 
of  the  variations  is  not  yet  established.  It  should  be  borne  in  mind 
in  this  connection  that  gas  volumes  are  frequently  variable  with  the 
same  indindual.  For  this  reason  it  is  necessary  in  every  instance  to 
follow  the  gas  production  for  a  considerable  period  of  time  before 
drawing  conclusions.^ 

For  diagnostic  purposes  the  macroscopical  and  microscopical 
examinations  of  the  feces  ordinarily  yield  much  more  satisfactory  data 
than  are  secured  from  its  chemical  examination. 


Experiments  ox  Feces. 


I.  Macroscopical  Examination. — If  the  stool  is  watery  pour 
it  into  a  shallow  dish  and  examine  directly.  If  it  is  firm  or  pasty 
it  should  be  treated  with  water  and  carefully  stirred  before  the 
examination  for  macroscopical  constituents  is  attempted. 

The  macroscopical  constituents  may  be  collected  very  satisfactorily 
by  means  of  a  Boas  sieve  (Fig.  49).  This  sieve  is  constructed  of 
two  easily  detachable  hemispheres  which  are  held 
together  by  means  of  a  bayonet  catch.  In  using 
the  apparatus  the  feces  is  spread  out  upon  a  very 
fine  sieve  contained  in  the  lower  hemisphere  and  a 
stream  of  water  is  allowed  to  play  upon  it  through 
the  medium  of  an  opening  in  the  upper  hemisphere. 
The  apparatus  is  provided  with  an  orifice  in  the 
upper  hemisphere  through  which  the  feces  may  be 
stirred  by  means  of  a  glass  rod  during  the  washing 
process.  After  15-30  minutes'  washing  nothing  but 
the  coarse  fecal  constituents  remain  upon  the  sieve. 

2.  Microscopical  Examination. — Watery  stools 
should  be  placed  in  a  shallow  dish,  thoroughly 
mixed,  and  a  small  amount  removed  to  a  slide  for 
examination.  Stools  of  a  firm  or  pasty  consistency 
should  be  rubbed  up  in  a  mortar  with  jjhysiological  salt  solution 
and  a  small  jjortion  of  the  resulting  mixture  transferred  to  a  slide 
for  examination.  In  normal  feces  look  for  food  particles,  bacteria, 
and  crystalline  bodies.  In  pathological  stools,  in  addition  to  these 
substances,  look  for  animal  parasites  and  pathological  products  of  the 
intestinal  wall.    See  Fig.  46,  jjage  168. 


Hcrler  and  Kenflall:  loc.  c'U. 


FECES.  173 

2.  Reaction. — Thoroughly  mix  the  feces  and  apply  moist  red  and 
blue  litmus  papers  to  the  surface.  If  the  stool  is  hard  it  should  be 
mixed  with  water  before  the  reaction  is  taken.  Examine  the  stool 
as  soon  after  defecation  as  is  convenient,  since  the  reaction  may  change 
very  rapidly.  The  reaction  of  the  normal  stools  of  adult  man  is  ordi- 
narily neutral  or  faintly  alkaline  to  litmus,  but  seldom  acid.  Infants' 
stools  are  generally  acid  in  reaction. 

4.  Starch. — If  any  imperfectly  cooked  starch-containing  food 
has  been  ingested  it  will  be  possible  to  detect  starch  granules  by  a 
microscopical  examination  of  the  feces.  If  the  granules  are  not 
detected  by  a  microscopical  examination,  the  feces  should  be  placed 
in  an  evaporating  dish  or  casserole  and  boiled  with  water  for  a  few 
minutes.  Filter  and  test  the  filtrate  by  the  iodine  test  in  the  usual 
way  (see  page  45). 

5.  Cholesterol  and  Fat. — Extract  the  dry  feces  with  ether  in 
a  Soxhlet  apparatus  (see  Fig.  126).  If  this  apparatus  is  not  available 
transfer  the  dry  feces  to  a  flask,  add  ether,  and  shake  frequently  for 
a  few  hours.  Filter  and  remove  the  ether  by  evaporation.  The 
residue  contains  cholesterol  and  the  mixed  fats  of  the  feces.  For 
every  gram  of  fat  add  about  i  1/2  gram  of  solid  potassium  hydroxide 
and  25  c.c.  of  95  per  cent  alcohol  and  boil  in  a  flask  on  a  water-bath 
for  one-half  hour,  maintaining  the  volume  of  alcohol  constant.  This 
alcoholic-potash  has  saponified  the  mixed  fats  and  we  now  have  a 
mixture  of  soaps  and  cholesterol.  Add  sodium  chloride,  in  substance^ 
to  the  mixture  and  extract  with  ether  to  dissolve  out  the  cholesterol. 
Remove  the  ether  by  evaporation  and  examine  the  residue  micro- 
scopically for  cholesterol  crystals.  Try  any  of  the  other  tests  for 
cholesterol  as  given  on  page  155. 

6.  Blood. — Undecomposed  blood  may  be  detected  macroscop- 
ically.  If  uncertain,  look  for  erythrocytes  under  the  microscope,  and 
spectroscopically  for  the  spectrum  of  oxyhasmoglobin  (see  Absorption 
Spectra,  Plate  I). 

In  case  the  blood  has  been  altered  or  is  present  in  minute  amount 
("occult  blood"),  and  cannot  be  detected  by  the  means  just  men- 
tioned, the  following  tests  may  be  tried: 

{a)  Aloin-turpentine  Test. — Mix  the  stool  very  thoroughly  and 
take  about  5  grams  of  the  mixture  for  the  test.  Reduce  this  sample 
to  a  semi-fluid  mass  by  means  of  distilled  water  and  extract  very 
thoroughly  with  an  equal  volume  of  ether  to  remove  any  fat  which 
may  be  present.  Now  treat  the  extracted  feces  with  one-third  its 
volume  of  glacial  acetic  acid  and  10  c.c.  of  ether  and  extract  very 


174  PHYSIOLOGICAL    CHEMISTRY. 

thoroughly  as  before.     The   acid-ether   extract   will   rise   to   the   top 
and  may  be  removed. 

Introduce  2-t,  c.c.  of  this  acid-ether  solution  into  a  test-tube, 
add  an  equal  volume  of  a  dilute  solution  of  aloin  in  70  per  cent  alcohol 
and  2-3  c.c.  of  ozonized  turpentine  and  shake  the  tube  gently.  If 
blood  is  present  the  entire  volume  of  fluid  ordinarily  becomes  pink 
and  finally  cherry-red.  In  some  instances  the  color  will  be  limited 
to  the  aloin  solution  which  sinks  to  the  bottom.  This  color  reaction 
should  occur  within  fifteen  minutes  in  order  to  indicate  a  positive 
test  for  blood,  since  the  aloin  will  turn  red  of  itself  if  allowed  to  stand 
for  a  longer  period.  The  color  is  ordinarily  light  yellow  in  a  negative 
test.  Hydrogen  peroxide  is  not  a  satisfactory  substitute  for  turpentine 
in  the  test. 

(b)  Weber^s  Guaiac  Test. — Mix  a  little  feces  with  30  per  cent 
acetic  acid  to  form  a  fluid  mass.  Transfer  to  a  test-tube  and  .extract 
with  ether.  If  blood  is  present  the  ether  will  assume  a  brownish-red 
color.  Filter  off  the  ether  extract  and  to  a  portion  of  the  filtrate  add 
an  alcoholic  solution  of  guaiac  (strength  about  i  :6o)/  drop  by  drop, 
until  the  fluid  becomes  turbid.  Now  add  hydrogen  peroxide  or  old 
turpentine.  In  the  presence  of  blood  a  blue  color  is  produced  (see 
page    191). 

(c)  Cowie's  Guaiac  Test. — To  i  gram  of  moist  feces  add  4-5  c.c. 
of  glacial  acetic  acid  and  extract  the  mixture  with  30  c.c.  of  ether. 
To  1-2  c.c.  of  the  extract  add  an  equal  volume  of  water,  agitate  the  mix- 
ture, introduce  a  few  granules  of  powdered  guaiac  resin,  and  after 
bringing  the  resin  into  solution,  gradually  add  30  drops  of  old  tur- 
pentine or  hydrogen  peroxide.  A  blue  color  indicates  the  presence 
of  blood.  Cowie  claims  that  by  means  of  this  test  an  intestinal  hem- 
orrhage of  I  gram  can  easily  l^e  detected  by  an  examination  of  the 
feces. 

(d)  Acid-hcBmatin. — Examine  some  of  the  ethereal  extract  from 
Experiment  (b)  spectroscopically.  Note  the  typical  s])ectrum  of 
acid-.hx'matin  (sec  Absorption  Spectra,  Plate  11). 

7.  Hydrobilirubin.  S(limidt\<;  Test. —  Rub  up  a  small  amount  of 
feces  in  a  mortar  with  a  concentrated  afpieous  solulioii  of  mercuric 
chloride.  Transfer  I0  a  shallow,  Hat-bottomed  dish  and  allow  to 
stand  6-24  hours.  The  presence  of  hydrobilirubin  will  be  indicated 
by  a  deep  red  color  being  imparted  to  the  particles  of  feces  containing 
this  pigment.    This  red  color  is  due  lo  (lie  formalioii  of  hydrobilirubin- 

'  Buckmastcr  advises  the  use  of  an  alcoholic  solution  of  gu.iiiironic  acid  instead  of  an 
alcoholic  srjlution  of  guaiac  resin. 


FECES.  175 

mercury.  If  unaltered  bilirubin  is  present  in  any  portion  of  the  feces 
that  portion  will  be  green  in  color  due  to  the  oxidation  of  bilirubin 
to  biliverdin. 

Another  method  for  the  detection  of  hydrobilirubin  is  the  following: 
Treat  the  dry  feces  with  absolute  alcohol  acidified  with  sulphuric  acid 
and  shake  thoroughly.  The  acidified  alcohol  extracts  the  pigment 
and  assumes  a  reddish  color.  Examine  a  little  of  this  fluid  spectro^ 
scopically  and  note  the  typical  spectrum  of  hydrobilirubin  (Absorption 
Spectra,  Plate  II). 

8.  Bilirubin/  (a)  Gmelin's  Test. — Place  a  few  drops  of  concen- 
trated nitric  acid  in  an  evaporating  dish  or  on  a  porcelain  test-tablet 
and  allow  a  few  drops  of  the  feces  and  water  to  mix  with  it.  The  usual 
play  of  colors  of  Gmelin's  test  is  produced,  i.  e.,  green,  blue,  violet, 
red,  and  yellow.  If  so  desired,  this  test  may  be  executed  on  a  slide  and 
observed  under  a  microscope. 

(b)  Hupperfs  Test. — Treat  the  feces  with  water  to  form  a  semi- 
fluid mass,  add  an  equal  amount  of  milk  of  lime,  shake  thoroughly, 
and  filter.  Wash  the  precipitate  with  water,  then  transfer  both  the 
paper  and  the  precipitate  to  a  small  beaker  or  flask,  add  a  small  amount 
of  95 'per  cent  alcohol  acidified  slightly  with  sulphuric  acid,  and  heat 
to  boiling  on  a  water-bath.  The  presence  of  bilirubin  is  indicated  by 
the  alcohol  assuming  a  green  color. 

Steensma  advises  the  addition  of  a  drop  of  a  0.5  per  cent  solution 
of  sodium  nitrite  to  the  acid-alcohol  mixture  before  warming  on  the 
water-bath.     Try  this  modification  also. 

9.  Bile  Acids. — Extract  a  small  amount  of  feces  with  alcohol 
and  filter.  Evaporate  the  filtrate  on  a  water-bath  to  drive  off  the  alcohol 
and  dissolve  the  residue  in  water  made  slightly  alkaline  with  potassium 
hydroxide.  Upon  this  aqueous  solution  try  any  of  the  tests  for  bile 
acids  'given  on  page  153. 

10.  Caseinogen. — Extract  the  fresh  feces  first  with  a  dilute  solu- 
tion of  sodium  chloride,  and  later  with  water  acidified  with  dilute 
acetic  acid,  to  remove  soluble  proteins.  Now  extract  the  feces  with 
0.5  per  cent  sodium  carbonate  and  filter.  Add  dilute  acetic  acid  to 
the  filtrate  to  precipitate  the  caseinogen,  being  careful  not  to  add  an 
excess  of  the  reagent  as  the  caseinogen  would  dissolve.  Filter  off  the 
caseinogen  and  test  it  according  to  directions  given  on  page  219.  Case- 
inogen is  found  principally  in  the  feces  of  children  who  have  been  fed 
a  milk  diet.     Mucin  would  also  be  extracted  by  the  dilute  alkali,  if 

*  The  detection  of  bilirubin  in  the  feces  is  comparatively  simple  provided  it  is  not 
accompanied  by  other  pigments.  When  other  pigments  are  present,  however,  it  is  difficult 
to  detect  the  bilirubin  and,  at  times,  may  be  found  impossible. 


176  PHYSIOLOGICAL   CHEMISTRY. 

present  in  the  feces.     What  test  could  you  make  on  the  newly  precipi- 
tated body  to  differentiate  between  mucin  and  caseinogen? 

11.  Nucleoprotein. — Mix  the  stool  thoroughly  with  water,  trans- 
fer to  a  flask,  and  add  an  equal  amount  of  saturated  lime  water. 
Shake  frequently  for  a  few  hours,  filter,  and  precipitate  the  nucleo- 
protein with  acetic  acid.  Filter  off  this  precipitate  and  test  it  as 
follows: 

(a)  Phosphorus. — Test  for  phosphorus  by  fusion  (see  page  247). 

(b)  Solubility. — Try  the  solubility  in  the  ordinary  solvents. 

(c)  Protein  Color  Test. — Try  any  of  the  protein  color  tests. 

What  proof  have  you  that  the  above  body  was  not  mucin  ?  What 
other  test  can  you  use  to  differentiate  between  nucleoprotein  and 
mucin  ? 

12.  Albumin  and  Globulin. — Extract  the  fresh  feces  with  a 
dilute  solution  of  sodium  chloride.  (The  preliminary  extract  from 
the  preparation  of  caseinogen  (10),  above,  may  be  utilized  here.) 
Filter,  and  saturate  a  portion  of  the  filtrate  with  sodium  chloride  in 
substance.  A  precipitate  signifies  globulin.  Filter  off  the  precipitate 
and  acidify  the  filtrate  slightly  with  dilute  acetic  acid.  A  precipitate 
at  this  point  signifies  albumin.  Make  a  protein  color  test  on  each  of 
these  bodies. 

13.  Proteose  and  Peptone. — Heat  to  boiling  the  portion  of  the 
sodium  chloride  extract  not  used  in  the  last  experiment.  Filter  off 
the  coagulum,  if  any  forms.  Acidify  the  filtrate  slightly  with  acetic 
acid  and  saturate  with  sodium  chloride  in  substance.  A  precipitate 
here  indicates  proteose.  Filter  it  off  and  test  it  .according  to  directions 
given  on  page  in.     Test  the  filtrate  for  peptone  by  the  biuret  test. 

14.  Inorganic  Constituents. ^Prepare  a  dilute  aqueous  solution 
of  dry  feces  and  decolorize  it  by  means  of  purified  animal  charcoal. 
Make  the  following  tests  upon  the  clear  solution. 

(a)  Chlorides. — Acidify  with  nitric  acid  and  add  argentic  nitrate. 

(b)  Phosphates. — Acidify  with  nitric  acid,  add  molybdic  solution, 
and  warm  gently. 

ic)  Sulphates. — Acidify  with  hydrochloric  acid,  add  barium  chloride, 
and  warm. 

15.  Konto's  Reaction  for  Indole.-  Rub  up  the  stool  with  water 
to  form  a  thin  jjaste.  From  this  point  the  test  is  the  same  as  for  the 
detection  of  indole  in  putrefaction  mixtures  (see  page  165). 

16.  Schmidt's  "Nuclei  Test. — This  test  serves  as  an  aid  to  the 
diagnosis  of  pancreatic  insufficiency.  The  test  is  founded  upon  the 
theory  that  cell  nuclei  are  digestible  only  in  pancreatic  juice,  and  there- 


FECES.  177 

fore  that  the  appearance  in  the  feces  of  such  nuclei  indicates  insuffi- 
ciency of  pancreatic  secretion.  The  procedure  is  as  follows:  Cubes 
of  fresh  beef  about  one-half  centimeter  square  are  enclosed  in  small 
gauze  bags  and  ingested  with  a  test  meal.  Subsequently  the  fecal 
mass  resulting  from  this  test-meal  is  examined,  the  bag  opened,  and 
the  condition  of  the  enclosed  residue  determined.  Under  normal 
conditions  the  nuclei  would  be  digested.  Therefore  if  the  nuclei  are 
found  to  be  for  the  most  part  undigested,  and  the  intervening  period 
has  been  sufficient  to  permit  of  the  full  activity  of  the  pancreatic  func- 
tion (at  least  six  hours),  it  may  be  considered  a  sign  of  pancreatic 
insufficiency. 

It  has  been  claimed  by  Steele  that  under  certain  conditions  the  non- 
digestion  of  the  nuclei  may  indicate  a  general  lowering  of  the  digestive 
power  rather  than  a  true  pancreatic  insufficiency. 


CHAPTER  XII. 
BLOOD. 

Blood  is  composed  of  four  types  of  form-elements  (erythrocytes 
or  red  blood  corpuscles,  leucocytes  or  white  blood  corpuscles,  blood 
plates  or  plaques  and  blood  dust  or  haemoconien)  held  in  suspension 
in  a  fluid  called  blood  plasma.  These  form-elements  compose  about 
60  per  cent  of  the  blood,  by  weight.  Ordinarily  blood  is  a  dark  red 
opaque  fluid  due  to  the  presence  of  the  red  blood  corpuscles,  but 
through  the  action  of  certain  substances,  such  as  water,  ether,  or  chloro- 
form, it  may  be  rendered  transparent.  Blood  so  altered  is  said  to  be 
laked.  The  laking  process  is  simply  a  liberation  of  the  haemoglobin 
from  the  stroma  of  the  red  blood  corpuscle.  Normal  blood  is  alkaline 
in  reaction^  to  litmus,  the  alkalinity  being  due  principally  to  sodium 
carbonate  and  phosphate.  The  specific  gravity  of  the  blood  of  adults 
ordinarily  varies  between  1.045  ^-nd  1.075.  ^^  varies  somewhat  with 
the  sex,  the  blood  of  males  having  a  rather  higher  specific  gravity 
than  that  of  females  of  the  same  species.  Under  pathological  con- 
ditions also  the  density  of  the  blood  may  be  very  greatly  altered.  The 
freezing-point  (J)  of  normal  blood  is  about  — 0.56°  C.  Variations 
between  —0.51°  and  0.62°  C.  may  be  due  entirely  to  dietary  conditions, 
but  if  any  marked  variation  is  noted  it  can  in  most  cases  be  traced  to 
a  disordered  kidney  function.  The  total  amount  of  blood  in 
the  body  has  been  variously  estimated  at  from  one-twelfth  to  one- 
fourteenth  of  the  body  weight.  Perhaps  1/13.5  is  the  most  satis- 
factory figure. 

Among  the  most  important  constituents  of  blood  plasma  are  the 
four  protein  bcxlies,  fibrinogen,  nucleo protein,  serum  globulin  (euglobu- 
lin  and  jjseudo-globulin)  and  serum  albumin.  Plasma  contains  about 
8.2  per  cent  of  solids  of  which  the  ])rotein  constituents  named  above 
constitute  approximately  84  per  cent  and  the  inorganic  constituents 
("mainly  chlorides,  phosphates  and  carbonates)  a]j])r()ximalcly  10  per 
cent.  Among  the  inorganic  constituents  sodium  chloride  ])redominates. 
To  prevent  coagulation,  blof)d  j)lusma  is  ordinarily  stu(li(;d  in  ihe  form 

'  Recently  it  has  been  shown  by  physi(:o-(:hcmi(  al  methods  tliat  ihc  Ijloorl  is  in  reality 
neutral  in  reaction. 

178 


BLOOD.  179 

of  an  oxalated  or  salted  plasma.  The  former  may  be  obtained  by 
allowing  the  blood  to  flow  from  an  opened  artery  into  an  equal  volume 
of  0.2  per  cent  ammonium  oxalate  solution,  whereas  in  the  preparation 
of  a  salted  plasma  10  per  cent  sodium  chloride  solution  may  be  used 
as  the  diluting  fluid. 

Fibrinogen  is  perhaps  the  most  important  of  the  protein  constituents 
of  the  plasma.  It  is  also  found  in  lymph  and  chyle  as  well  as  in  cer- 
tain exudates  and  transudates.  Fibrinogen  possesses  the  general 
properties  of  the  globulins,  but  differs  from  serum  globulin  in  being 
precipitated  upon  half-saturation  with  sodium  chloride.  In  the  proc- 
ess of  coagulation  of  the  blood  the  fibrinogen  is  transformed  into 
fibrin.  This  fibrin  is  one  of  the  principal  constituents  of  the  ordinary 
blood  clot. 

The  nucleoprotein  of  blood  possesses  many  of  the  characteristics 
of  serum  globulin.  In  common  with  this  body  it  is  easily  soluble  in 
sodium  chloride,  and  is  completely  precipitated  from  its  solutions  upon 
saturation  with  magnesium  sulphate.  It  is  much  less  soluble  in  dilute 
acetic  acid  than  serum  globulin,  and  its  solutions  coagulate  at  65°-69°  C. 

The  body  formerly  called  serum  globulin  is  probably  not  an  indi- 
vidual substance.  Recent  investigations  seem  to  indicate  that  it  may 
be  resolved  into  two  individual  bodies  called  euglohulin  and  pseudo- 
globulin.  The  euglobulin  is  practically  insoluble  in  water  and  may 
be  precipitated  in  the  presence  of  28-36  per  cent  of  saturated  ammo- 
nium sulphate  solution.  The  pseudoglobulin,  on  the  contrary,  is 
soluble  in  water  and  is  only  precipitated  by  ammonium  sulphate  in 
the  presence  of  from  36  to  44  per  cent  of  saturated  ammonium  sulphate 
solution. 

In  common  with  serum  globulin  the  body  known  as  serum  albu- 
min seems  also  to  consist  of  more  than  a  single  individual  substance. 
The  so-called  serum  albumin  may  be  separated  into  at  least  two  dis- 
tinct bodies,  one  capable  of  crystallization,  the  other  an  amorphous 
body.  The  solution  of  either  of  these  bodies  in  water  gives  the  ordi- 
nary albumin  reactions.  The  coagulation  temperature  of  the  serum 
albumin  mixture  as  it  occurs  in  serum  or  plasma  varies  from  70°  to  85° 
C.  according  to  the  reaction  of  the  solution  and  its  content  of  inorganic 
material.  Serum  albumin  differs  from  egg  albumin  in  being  more 
lasvorotatory,  in  being  rendered  less  insoluble  by  alcohol,  and  in  the 
fact  that  when  precipitated  by  hydrochloric  acid  it  is  more  easily  solu- 
ble in  an  excess  of  the  reagent. 

When  blood  coagulates  and  the  usual  clot  forms,  a  light  yellow 
fluid  exudes.     This  is  blood   serum.     It  differs   from  blood  plasma 


I  So  PHYSIOLOGICAL    CHEMISTRY. 

in  containing  a  large  amount  oi  fibrin  ferment,  a  body  of  great  impor- 
tance in  the  coagulation  of  the  blood,  and  also  in  possessing  a  lower  pro- 
tein content.  The  protein  material  present  in  plasma  and  not  found 
in  serum  is  the  fibrinogen  which  is  transformed  into  fibrin  in  the  proc- 
ess of  coagulation  and  removed.  The  specific  gravity  of  the  serum 
of  human  blood  varies  between  1.026  and  1.032.  If  blood  be  drawn 
into  a  vessel  and  allowed  to  remain  without  stirring  or  agitation  of  any 
sort  the  major  portion  of  the  red  corpuscles  will  sink  away  from  the 
upper  surface,  causing  this  portion  of  the  clot  to  assume  a  lighter  color 
due  to  the  predominance  of  leucocytes.  This  light  colored  portion  of 
the  clot  is  called  the  "buffy  coat." 

Beside  the  protein  constituents  already  mentioned,  other  bodies 
which  are  found  in  both  the  plasma  and  serum  are  the  following: 
Sugar  (dextrose), /<3/,,  enzymes,  lecithin ,  cholesterol  and  its  esters,  gases, 
coloring-matter  (lutein  or  lipochrome)  and  mineral  substances.  In 
addition  to  these  bodies  the  following  substances  have  been  detected 
in  normal  human  blood:  Creatine,  carbamic  acid,  hippuric  acid,  para- 
lactic  acid,  urea  and  uric  acid  {urates).  Some  of  the  pathological  can- 
stitnents  of  blood  are  proteoses,  leucine,  tyrosine  and  other  amino 
acids,  biliary  canstituents  and  purine  bodies. 

There  has  recently  been  considerable  controversy  regarding  the 
form  of  the  erythrocytes  or  red  blood  corpuscles  of  human  blood.  It 
is  claimed  by  some  investigators  that  the  cells  are  bell-shaped  or  cup- 
shaped.  As  the  erythrocytes  occur  normally  in  the  circulation,  how- 
ever, they  are  probably  thin,  non-nucleated,  biconcave  discs.  When 
examined  singly  under  the  microscope,  they  possess  a  pale  greenish- 
yellow  color  (see  Plate  IV,  opposite),  whereas  when  grouped  in  large 
masses  a  reddish  tint  is  noted. 

The  blood  of  most  mammals  contains  erythrocytes  similar  in  form 
to  those  of  human  blood.  In  the  blood  of  birds,  fishes,  amphibians 
and  reptiles  the  erythrocytes  are  ordinarily  more  or  less  elliptical,  bicon- 
\'ex  and  possess  a  nucleus.  The  erythrocytes  vary  in  size  with  the 
dilTerent  animals.  The  average  diameter  of  the  erythrocytes  of  blood 
irum  \arious  species  is  gi\'en  in  the  following  lal)lc:* 

Klcphanl ■•■r,\H  "f  ''"  'i"  'i- 

(iujnea-pi^    na'aa  "^  =1"  '"^'i' 

Man ;)  ..'nn  "<"  ^i"  ''"" 'i- 

Monkey -jm's-  "f  ■'n  i"<  I'- 

Dog    ;, ,-,',1 1  "f  'III  i'"-''- 

Rat :i I',',-, L'  t)^  ''"  iiK  li. 

'  Wormley's  Micro-Chemistry  uf  I'oisoiis,  se(niiil  ((liiion,  \>.  7  .j.:;. 


PLATE  IV. 


Normal  Erythrocytes  and  Leucocytes. 


.  BLOOD.  151 

Rabbit   s 8^53  of  an  inch. 

Mouse    Tjris  of  an  inch. 

Lion j:t?3  of  ^^  inch. 

Ox lAir  of  an  inch. 

Horse ¥5^?3  of  an  inch. 

Pig    fSaH  of  an  inch. 

Cat     ?3Ti5  of  an  inch. 

Sheep 47rT2  of  an  inch. 

Goat 6tV;i  of  an  inch. 

Alusk-deer , 123 a.-,  of  an  inch. 

The  erythrocytes  from  whatever  source  obtained,  consist  essen- 
tially of  two  parts,  the  stroma  or  protoplasmic  tissue  and  its  enclosed 
pigment,  hamoglobin.  For  human  blood  the  number  of  erythrocytes 
present  in  the  fluid  as  obtained  from  well-developed  males  in  good 
physical  condition  is  about  5,500,000  per  cubic  millimeter.^  The 
normal  content  of  the  blood  of  adult  females  is  from  4,000,000  to 
4,500,000  per  cubic  millimeter.  The  number  of  erythrocytes  varies 
greatly  under  different  conditions.  For  instance  the  number  may  be 
increased  after  the  transfusion  of  blood  of  the  same  species  of  animal; 
by  residing  in  a  high  altitude;  or  as  a  result  of  strenuous  physical  exer- 
cise continued  over  a  short  period  of  time.  An  increase  is  also  noted 
in  starvation;  after  partaking  of  food;  after  cold  or  hot  baths;  after 
massage,  as  well  as  after  the  administration  of  certain  drugs  and  accom- 
panying certain  diseases,  such  as  cholera,  diarrhoea,  dysentery  and 
yellow  atrophy  of  the  liver.  A  decrease  in  the  number  occurs  in  the 
different  forms  of  anaemia.  The  number  has  been  known  to  increase 
to  7,040,000  per  cubic  millimeter  as  a  result  of  physical  exercise,  while 
11,000,000  per  cubic  millimeter  have  been  noted  in  cases  of  polycythae- 
mia  and  increases  nearly  as  great  in  cyanosis.  The  number  has  been 
known  to  decrease  to  500,000  per  cubic  millimeter  or  lower  in  per- 
nicious anaemia. 

Oxyhaemoglobin,  the  coloring  matter  of  the  blood,  is  a  conjugated 
protein.  Through  treatment  with  hydrochloric  acid  it  may  be  split 
into  a  protein  body  called  globin,  and  hamochromogen,  an  iron-con- 
taining pigment.  The  latter  body  is  rapidly  transformed  into  hamatin 
in  the  presence  of  oxygen,  and  this  in  turn  gives  place  to  haematin- 
hydrochloride  or  hcBmin  (Figs.  58  and  59,  page  194).  The  pigment  of 
arterial  blood  is  for  the  most  part  loosely  combined  with  oxygen  and  is 
termed  o.Tyhcemoglobin,  whereas  the  pigment  of  venous  blood  is  prin- 
cipally haemoglobin  (so-called  reduced  haemoglobin).     Oxyhaemoglobin 

'  This  statement  is  based  upon  observations  made  upon  the  blood  of  athletes  in  training-. 
It  is  generally  stated  in  text-books  that  the  blood  of  males  contains  about  5,000,000  per 
cubic  millimeter. 


l82 


PHYSIOLOGICAL   CHEMISTRY. 


Fig.  50. — OwTi-EMOGLOBiN  Crystals  from  Blood  of  the  Guinea-pig. 
Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the  University 

of  Pennsylvania. 


V;*' 


Fig.  51. — r).\vn.K.vi(K;L<JiJi.v  Ckystals  fuom  JIi.odd  on   iiik  I\\i. 
Reprodut:cfi  from  a  mif  ro-jjholograph  furnislied  by  I'rf)f.  K.  '1'.  Rcii  luil,  of  the  IniviTsiiy 

of  I'crifisylv.inia. 


BLOOD. 


183 


Fig.  52. — OXYH.EMOGLOBIN  Crystals  from  Blood  of  the  Horse. 
Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the  University 

of  Pennsylvania. 


Fig.  53. — OxYH.EMOGLOBiN  Crystals  from  Blood  of  the  Squirrel. 
Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the  University 

of  Pennsylvania. 


iS4 


PHYSIOLOGICAL   CHEMISTRY. 


=*^ 


Fig.  54. — OxvH.EiioGLOBix  Crystals  from  blood  of  the  Dog. 
Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the  University 

of  Pennsylvania. 


Fig.  55. — OxYH>E.\ioGLOHL\  Crystals  ikom  lU.onD  (>\-   ihk  Cai, 
Reproduced  from  a  micro-photograph  furnished  by  I'rof.  Iv  'I".  I<ci(  licit,  of  llu-  I  irncTsiU' 

of  I'cnnsylvania. 


BLOOD. 


Fig.  56. — O.w'H.EMOGLOBix  Cryst.^ls  from  Blood  of  the  Xecturus. 
Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the  University 

of  Pennsylvania.  '■ 


is  the  oxygen-carrier  of  the  body  and  belongs  to  the  class  of  bodies 
known  as  respiratory  pigments.  It  is  held  within  the  stroma  of  the 
erythrocyte.  The  reduction  of  oxyhaemoglobin  to  form  haemoglobin 
(so-called  reduced  haemoglobin)  occurs  in  the  capillaries.  Oxyhcemo- 
globin  may  be  crystallized  and  a  specific  form  of  crystal  obtained 
from  the  blood  of  each  individual  species  (see  Figs.  50  to  56,  pages  182 
to  185).  This  fact  seems  to  indicate  that  there  are  many  varieties  of 
oxyhaemoglobin.  The  interesting  findings  of  Reichert  and  Brown  are 
of  great  value  in  this  connection.  These  investigators  prepared  oxy- 
haemoglobin crystals  from  o^•er  one  hundred  species  of  animal  and  sub- 
sequently studied  the  characteristics  of  the  crystals  very  minutely 
from  the  standpoint  of  crystallography.  Their  findings  may  prove  of 
importance  from  the  standpoint  of  heredity  and  the  origin  of  species. 
They  emphasize  the  following  facts: 

1.  Crystals  from  all  species  of  a  certain  genus  have  certain  charac- 
teristics in  general.  Crystals  from  dift'erent  genera,  however,  exhibit 
marked  differences  in  system,  axial  ratios,  etc. 

2.  Crystals  of  different  species  of  a  genus  may  generally  be  dift'eren- 
tiated  by  difference  in  the  angles. 

3.  The  oxyhaemoglobin  of  some  species  crystallizes  in  several  types 
of  crystals  in  the  same  preparation.  Generally  the  crystals  first  formed 
belong  to  a  system  of  a  lower  grade  of  symmetry  than  those  formed 

'  The  micro-photographs  of  oxyhsemoglobin  (see  pages  1S2-1S5)  a-^^d  ha^min  (see 
page  194)  are  reproduced  through  the  courtesy  of  Professors  E.  T.  Reichert  and  Amos 
P.  Brown,  of  the  University  of  Pennsylvania,  who  are  investigating  the  cr\'stalline  forms 
of;,biochemic  sulistances. 


1 86  PHYSIOLOGICAL   CHEMISTRY. 

later.     WTien  such  different  types  of  crystals  occur  they  may  be  arranged 
in  isomorphous  series. 

4.  Certain  definite  angles  recur  in  the  crystals  from  the  blood  of 
various  species  of  animal,  although  the  zoological  connection  may  be 
remote  and  the  crystals  belong  to  different  systems. 

5.  The  constant  recurrence  of  certain  types  of  "twinning"  in  all 
the  crystalline  forms  was  observed. 

6.  Differences  have  been  observed  in  the  crystalline  form  of  oxy- 
haemoglobin  and  haemoglobin  from  the  blood  of  the  same  species  in 
certain  cases. 

The  following  bodies  may  be  derived  from  haemoglobin,  and  each 
possesses  a  specific  spectrum  which  serves  as  an  aid  in  its  detection 
and  identification:  Oxy haemoglobin,  methaemoglobin,  carbon-mon- 
oxide haemoglobin,  nitric-oxide  haemoglobin,  haemochromogen,  haema- 
tin,  acid-haematin,  alkali-haematin  and  haematoporphyrin  (see  Absorp- 
tion Spectra,  Plates  I  and  II). 

The  white  corpuscles  (or  leucocytes)  of  human  blood  differ  from 
the  red  corpuscles  (or  erythrocytes)  in  many  particulars,  such  as  being 
somewhat  larger  in  size,  in  containing  at  least  a  single  nucleus  and  in 
possessing  amoeboid  movement  (see  Plate  IV,  opposite  page  180). 
They  are  typical  animal  cells  and  therefore  contain  the  following  bodies 
which  are  customarily  present  in  such  cells:  Proteins,  fats,  carbohy- 
drates, lecithin,  cholesterol,  inorganic  salts  and  water.  The  normal 
number  of  leucocytes  in  human  blood  varies  between  5,000  and  10,000 
per  cubic  millimeter.  The  ratio  between  the  leucocytes  and  erythro- 
cytes is  about  1:350-500.  A  leucocytosis  is  said  to  exist  when  the 
number  of  leucocytes  is  increased  for  any  reason.  Leucocytoses  may 
be  divided  into  two  general  classes,  the  physiological  and  the  patho- 
logical. Under  the  physiological  form  would  be  classed  those  leuco- 
cytoses accompanying  pregnancy,  ])arturilion  and  digestion,  as  well 
as  those  due  to  mechanical  and  thermal  influences.  The  leucocytoses 
spoken  of  as  pathological  are  the  inflammatory,  infectious,  post-haemor- 
rhagic,  toxic  and  experimental  forms  as  well  as  the  type  of  leucocytosis 
which  accompanies  malignant  disease. 

The  blood  plates  (j)latelets  or  placjues)  arc  round  or  oval,  colorless 
discs  which  possess  a  diameter  about  one-third  as  great  as  that  of  the 
erythrocytes.  Upon  treatment  with  certain  reagents,  e.  g.,  artificial 
gastric  juice,  they  may  be  separated  into  a  homogeneous,  non-refractive 
portion  and  a  granular,  refractive  yjortion.  The  blood  plates  arc 
probably  associated  in  some  way  with  the  coagulation  of  the  ]>lood. 
This  relationship  is  not  well  understood  at  ])resent. 


BLOOD.  187 

The  hgemoconein  or  so-called  "blood  dust"  is  made  up  of  round 
granules  which  usually  have  a  diameter  somewhat  less  than  one  micron. 
The  serum  of  normal  as  well  as  of  pathological  blood  contains  these 
granules.  They  were  first  described  by  Miiller  to  whom  they  appeared 
as  highly  refractile  granules  possessed  of  Brownian  movement.  The 
"blood  dust"  is  apparently  not  concerned  with  the  coagulation  of  the 
blood.  The  granules  are  insoluble  in  alcohol,  ether  and  acetic  acid 
and  are  not  blackened  by  osmic  acid.  According  to  Miiller,  the  gran- 
ules making  up  the  so-called  "blood  dust"  constitute  a  new  organized 
constituent  of  the  blood,  whereas  other  investigators  believe  them  to  be 
merely  free  granules  from  certain  of  the  forms  of  leucocytes.  In 
common  with  blood  plates  the  "blood  dust"  possesses  no  clinical 
significance. 

The  processes  involved  in  the  coagulation  of  the  blood  are  not 
fully  understood.  Several  theories  have  been  advanced  and  each  has 
its  adherents.  The  theory  which  appears  to  be  fully  as  firmly  founded 
upon  experimental  evidence  as  any  is  the  following:  Blood  contains 
a  zymogen  called  prothrombin  which  combines  with  the  calcium  salts 
present  to  form  an  enzyme  known  as  thrombin  or  fibrin-ferment.  When 
freshly  drawn  blood  comes  in  contact  with  the  air  the  fibrin-ferment  at 
once  acts  upon  the  fibrinogen  present  and  gives  rise  to  the  formation 
oi  fibrin.  This  fibrin  forms  in  shreds  throughout  the  blood  mass  and, 
holding  the  form  elements  of  the  blood  within  its  meshes,  serves  to 
produce  the  typical  blood  clot.  The  fibrin  shreds  gradually  contract, 
the  whole  clot  assumes  a  jelly-like  appearance  and  the  yellowish  serum 
exudes.  If,  immediately  upon  the  withdrawal  of  blood  from  the  body, 
the  fluid  be  rapidly  stirred  or  thoroughly  "whipped"  w^ith  a  bundle 
of  coarse  strings,  twigs  or  a  specially  constructed  beater,  the  fibrin 
shreds  will  not  form  in  a  network  throughout  the  blood  mass  but 
instead  will  cling  to  the  device  used  in  beating.  In  this  way  the  fibrin 
may  be  removed  and  the  remaining  fluid  is  termed  defibrinated  blood. 
The  above  theory  of  the  coagulation  of  the  blood  may  be  stated  briefly 
as  follows : 

I.  Prothrombin  -|-  Calcium  Salts  =  Thrombin  (or  Fibrin-ferment). 

II.  Thrombin  (or  Fibrin-ferment)  +  Fibrinogen  =  Fibrin. 
Among  the  medico-legal  tests  for  blood  are  the  following:      (i) 

Microscopical  identification  of  the  erythrocytes,  (2)  spectroscopic 
identification  of  blood  solutions,  (3)  the  guaiac  test,  ("4)  the  benzidine 
reaction,  (5)  preparation  of  haemin  crystals.  Of  these  five  tests  the 
two  last  named  are  generally  considered  to  be  the  most  satisfactory. 
They  give  equally  reliable  results  with  fresh  blood  and  with  blood  from 


1 88  PHYSIOLOGICAL    CHEMISTRY. 

clots  or  Stains  of  long  standing,  provided  the  latter  have  not  been 
exposed  to  a  high  temperature,  or  to  the  rays  of  the  sun  for  a  long 
period.  The  technique  of  the  tests  is  simple  and  the  formation  of  the 
dark  brown  or  chocolate  colored  crystals  of  hasmin  or  the  production 
of  the  green  or  blue  color  with  benzidine  is  indisputable  proof  of  the 
presence  of  blood  in  the  fluid,  clot  or  stain  examined.  The  weak  point 
of  the  tests,  medico-legally,  lies  in  the  fact  that  they  do  not  differen- 
tiate between  human  blood  and  that  of  certain  other  species  of  animal. 

The  guaiac  test  (see  page  191),  although  generally  considered  less 
accurate  than  the  haemin  test,  is  really  a  more  delicate  test  than  the  haemin 
test  if  properly  performed.  One  of  the  most  common  mistakes  in  the 
manipulation  of  this  test  is  the  use  of  a  guaiac  solution  which  is  too 
concentrated  and  which,  when  brought  into  contact  with  the  aqueous 
blood  solution,  causes  the  separation  of  a  voluminous  precipitate  of  a 
resinous  material  which  may  obscure  the  blue  coloration:  this  is  par- 
ticularly true  of  the  test  when  used  for  the  examination  of  blood  stains. 
A  solution  of  guaiac  made  by  dissolving  i  gram  of  the  resin  in  60  c.c. 
of  95  per  cent  alcohol  is  very  satisfactory  for  general  use.  The  test 
is  frequently  objected  to  upon  the  ground  that  various  other  substances, 
e.  g.,  milk,  pus,  saliva,  etc.,  respond  to  the  test  and  that  it  cannot  there- 
fore be  considered  a  specific  test  for  blood  and  is  of  value  only  in  a 
negative  sense.  We  have  demonstrated  to  our  own  satisfaction,  how- 
ever, that  milk  many  times  gives  the  blue  color  upon  the  addition  of  an 
alcoholic  solution  of  guaiac  resin  without  the  addition  of  hydrogen 
peroxide  or  old  turpentine.  Buckmaster  has  very  recently  advocated 
the  use  of  an  alcoholic  solution  of  guaiaconic  acid  instead  of  an  alco- 
holic solution  of  guaiac  resin.  He  claims  that  he  was  able  to  produce 
the  blue  color  upon  the  addition  of  the  guaiaconic  acid  to  milk^only 
when  the  sample  of  milk  tested  was  brought  from  the  country  in  sterile 
hollies,  and  further,  that  no  sample  of  London  milk  which  he  examined 
responded  to  the  test.  In  the  application  of  the  guaiac  test  to  the  detec- 
tion of  blood,  he  states  that  he  was  able  to  detect  laked  blood  when 
present  in  the  ratio  1:5,000,000  and  tinlaked  blood  when  present  in  the 
ratio  1:1,000,000.  This  author  considers  the  guaiac  test  to  be  far 
more  trustworthy  than  is  generally  believed. 

Up  to  within  very  recent  times  it  has  been  impossible  to  make  an 
absolute  differentiation  of  human  blood.  Recently,  however,  the  so- 
called  "biological"  blood  test  has  made  such  a  differentiation  jjossible. 
This  test,  known  as  the  Bordet  reaction,  is  founded  upon  the  fact 
that  the  blood  serum  of  an  animal  into  which  has  been  injected  the 
blood  of  another  animal  of  different  species  develops  the  property  of 


BLOOD.  189 

agglutinating  and  dissolving  erythrocytes  similar  to  those  injected,  but 
exerts  this  influence  upon  the  blood  from  no  other  species.  The  anti- 
serum used  in  this  test  is  prepared  by  injecting  rabbits  with  5-10  c.c. 
of  human  defibrinated  blood,  at  intervals  of  about  four  days  until  a 
total  of  between  50  and  80  c.c.  has  been  injected.  After  a  lapse  of  one 
or  two  weeks  the  animal  is  bled,  the  serum  collected,  placed  in  sterile 
tubes  and  preserved  for  use  as  needed.  In  examining  any  specific 
solution  for  human  blood  it  is  simply  necessary  to  combine  the  anti- 
serum and  the  solution  under  examination  in  the  proportion  of  1:100 
and  place  the  mixture  at  37°  C.  If  human  blood  is  present  in  the  solu- 
tion a  turbidity  will  be  noted  and  this  will  change  within  three  hours 
to  a  distinctly  flocculent  precipitate.  This  antiserum  will  react  thus 
with  no  other  known  substance. 

Experiments  on  Blood. 

I.  Defibrinated  Ox-blood. 

1.  Reaction. — Moisten  red  and  blue  litmus  papers  with  10  per 
cent  sodium  chloride  solution  and  test  the  reaction  of  the  defibrinated 
blood. 

2.  Microscopical  Examination. — Examine  a  drop  of  defibri- 
nated blood  under  the  microscope.  Compare  the  objects  you  observe 
with  Plate  IV,  opposite  page  180.  Repeat  the  test  with  a  drop  of  your 
own  blood. 

3.  Specific  Gravity. — Determine  the  specific  gravity  of  defibri- 
nated blood  by  means  of  an  ordinary  specific  gravity  spindle.  Com- 
pare this  result  with  the  specific  gravity  as  determined  by  Hammer- 
schlag's  method  in  the  next  experiment. 

4.  Specific  Gravity  by  Hammers chlag's  Method. — Fill  an  ordi- 
nary urinometer  cylinder  about  one-half  full  of  a  mixture  of  chloro- 
form and  benzene,  having  a  specific  gravity  of  approximately  1.050. 
Into  this  mixture  allow  a  drop  of  the  blood  under  examination  to  fall 
from  a  pipette  or  directly  from  the  finger  in  case  fresh  blood  is  being 
examined.  Care  must  be  taken  not  to  use  too  large  a  drop  of  blood 
and  to  keep  the  drop  from  coming  in  contact  with  the  walls  of  the 
cylinder.  If  the  blood  drop  sinks  to  the  bottom  of  the  vessel,  thus 
showing  it  to  be  of  higher  specific  gravity  than  the  surrounding  fluid, 
add  chloroform  until  the  blood  drop  remains  suspended  in  the  mix- 
ture. Stir  carefully  with  a  glass  rod  after  adding  the  chloroform.  If 
the  blood  drop  rises  to  the  surface  upon  being  introduced  into  the  mix- 
ture, thus  showing  it  to  be  of  lower  specific  gravity  than  the  surrounding 


ipO  PHYSIOLOGICAL   CHEMISTRY. 

fluid,  add  benzene  until  the  blood  drop  remains  suspended  in  the 
mixture.  Stir  with  a  glass  rod  after  the  benzene  is  added.  After  the 
blood  drop  has  been  brought  to  a  suspended  position  in  the  mixture 
by  means  of  one  or  more  additions  of  chloroform  and  benzene  this 
final  mixture  should  be  filtered  through  muslin  and  its  specific 
gra\ity  accurately  determined.  What  is  the  specific  gravity  of  the 
blood  under  examination  ? 

5.  Tests  for  Various  Constituents. — Place  10  c.c.  of  defibri- 
nated  blood  in  an  evaporating  dish,  dilute  with  100  c.c.  of  water  and 
heat  to  boiling.  Is  there  any  coagulation,  and  if  so  what  bodies  form 
the  coagulum?  At  the  boiling-point  acidulate  slightly  with  dilute 
acetic  acid.  Filter.  The  filtrate  should  be  clear  and  the  coagulum 
dark  brown.  Reserve  this  coagulum.  What  body  gives  the  coagu- 
lum this  color?  Evaporate  the  filtrate  to  about  25  c.c,  filtering  off 
any  precipitate  which  may  form  in  the  process.  Make  the  following 
tests  upon  the  filtrate : 

(a)  Fehling's  Test. — Test  for  sugar  according  to  directions  given 
on  page  27. 

(6)  Chlorides. — To  a  small  amount  of  the  filtrate  in  a  test-tube  add 
a  few  drops  of  nitric  acid  and  a  little  argentic  nitrate.  In  the  presence 
of  chloride,  a  white  precipitate  of  argentic  chloride  will  form. 

(c)  Phosphates. — Test  for  phosphates  by  nitric  acid  and  molybdic 
solution  according  to  directions  given  on  page  56. 

{d)  Proteose  and  Peptone. — Test  a  small  amount  of  the  solution 
for  proteose  and  peptone  by  saturating  with  ammonium  sulphate 
according  to  directions  given  on  page  112. 

(e)  Crystallization  of  Sodium  Chloride. — Place  the  remainder  of 
the  filtrate  in  a  watch  glass  and  evaporate  it  on  a  water-bath.  Examine 
the  crystals  under  the  microscope  and  compare  them  with  those  in 
Fig.  60,  page  196. 

6.  Test  for  Iron. — Incinerate  a  small  portion  of  the  coagulum 
from  the  last  experiment  (5)  in  a  porcelain  crucible.  Cool,  dissolve 
the  residue  in  dilute  hydrochloric  acid  and  test  for  iron  by  potassium 
ferrocyanide  or  ammonium  thiocyanate.  Which  of  the  constituents 
of  the  blof)d  contains  the  iron? 

7.  Laky  Blood. — Note  the  opacity  of  ordinary  dclibrinaled 
blood.  Place  a  few  cul)ic  centimeters  of  this  blood  in  a  test-tube 
and  add  water,  a  h"ltlc  at  a  lime,  until  the  blood  is  rendered  trans- 
jjarent.  It  is  now  laky  blood.  How  does  the  water  act  in  causing 
this  transy)arency  ?  lOxamine  a  droj)  of  laky  blood  under  the  micro- 
scope,    flow  does  its  microscopical   ap|>earance  differ   from   that   of 


BLOOD.  191 

unaltered  blood?     What  other  agents  may  be  used  to  render  blood 
laky? 

8.  Osmotic  Pressure. — Place  a  few  cubic  centimeters  of  blood 
in  each  of  three  test-tubes.  Lake  the  blood  in  the  first  tube  accord- 
ing to  directions  given  in  the  last  experiment  (7) :  add  an  equal  volume 
of  isotonic  (0.9  per  cent)  sodium  chloride  to  the  blood  in  the  second 
tube,  and  an  equal  volume  of  10  per  cent  sodium  chloride  to  the  blood 
in  the  third  tube.  Mix  thoroughly  by  shaking  and  after  a  few  mo- 
ments examine  a  drop  from  each  of  the  three  tubes  under  the  micro- 
scope (see  Figs.  57  and  115,  pages  191  and  354).  What  do  you  find 
and  what  is  your  explanation  from  the  standpoint  of  osmotic  pressure  ? 


Fig.  57. — Effect  of  Water  ox  Erythrocytes. 

9.  Agglutination. — To  about  5  c.c.  of  a  dilute  sodium  chloride 
solution  of  ricin^  in  a  test-tube  add  about  one-half  cubic  centimeter 
of  defibrinated  blood  and  shake  the  mixture  thoroughly.  Allow  the 
tube  to  stand  about  15  minutes  and  examine  a  drop  of  the  contents 
under  the  microscope.  Note  the  "clumping"  or  "agglutination" 
of  the  erythrocytes,  and  contrast  this  phenomena  with  the  appear- 
ance of  normal  blood  as  just  examined  in  Experiment  8. 

10.  Diffusion  of  Hasmoglobin. — Prepare  some  laky  blood,  thus 
liberating  the  haemoglobin  from  the  erythrocytes.  Test  the  dif- 
fusion of  the  haemoglobin  by  preparing  a  dialyzer  like  one  of  the  models 
shown  in  Fig.  i,  page  25.  How  does  haemoglobin  differ  from  other 
well-known  crystallizable  bodies? 

11.  Guaiac  Test. — To  5  c.c.  of  water  in  a  test-tube  add  two 
drops  of  blood.     By  means  of  a  pipette  drop  an  alcoholic  solution 

'  A  protein  constituent  of  the  castor  bean. 


192  PHYSIOLOGICAL    CHEMISTRY. 

of  guaiac  (strength  about  1:60)^  into  the  resuhing  mixture  until 
a  turbidity  is  observed  and  add  old  turpentine  or  hydrogen  peroxide, 
drop  by  drop,  until  a  blue  color  is  obtained.  Do  any  other  substances 
respond  in  a  similar  manner  to  this  test?  Is  a  positive  guaiac  test 
a  sure  indication  of  the  presence  of  blood  ? 

12.  Schumm's  Modification  of  the  Guaiac  Test. — To  about 
5  c.c.  of  the  solution  under  examination'-^  in  a  test-tube  add  about 
ten  drops  of  freshly  prepared  alcoholic  solution  of  guaiac.  Agitate 
the  tube  gently,  add  about  20  drops  of  old  turpentine,  subject  the 
tube  to  a  thorough  shaking  and  permit  it  to  stand  for  about  2-3 
minutes.  A  blue  color  indicates  the  presence  of  blood  in  the  solution 
under  examination.  In  case  there  is  insufficient  blood  to  yield  a  blue 
color  under  these  conditions,  a  few  c.c.  of  alcohol  should  be  added  and 
the  tube  gently  shaken,  whereupon  a  blue  coloration  will  appear  in  the 
upper  alcohol-turpentine  layer. 

A  control  test  should  always  be  made,  using  water  in  place  of  the 
solution  under  examination.  In  the  detection  of  very  minute  traces 
of  blood  only  3-5  drops  of  the  guaiac  solution  should  be  employed. 

13.  Adler's  Benzidine  Reaction. — This  is  one  of  the  most  deli- 
cate of  the  reactions  for  the  detection  of  blood.  Different  benzi- 
dine preparations  vary  greatly  in  their  sensitiveness,  however.  Inas- 
much as  benzidine  solutions  change  readily  upon  contact  with  light 
it  is  essential  that  they  be  kept  in  a  dark  place.  The  test  is  performed 
as  follows:  To  a  saturated  solution  of  benzidine  in  alcohol  or  glacial 
acetic  acid  add  an  equal  volume  of  3  per  cent  hydrogen  peroxide  and 
one  c.c.  of  the  solution  under  examination.  If  the  mixture  is  not 
already  acid  render  it  so  with  acetic  acid,  and  note  the  appearance  of 
a  green  or  blue  color.  A  control  test  should  be  made  substituting  water 
for  the  solution  under  examination.  The  sensitiveness  of  the  benzidine 
reaction  is  greater  when  applied  to  aqueous  solutions  than  when 
applied  to  the  urine.  According  to  Ascarclli^  the  benzidine  reaction 
serves  to  detect  blood  when  present  in  a  dilution  of  i :  300,000.  Walter' 
has  also  recently  shown  the  test  to  be  very  delicate  and  claims  it  to  be 
more  satisfactory  than  the  guaiac  test. 

14.  Haemin  Test. — (a)  Teichmann' s  Method. — Place  a  very  small 
drop  of  blood  on  a  microscopic  slide,  add  a  minute  grain  of  sodium 

*  Buckmastcr  advises  the  use  of  ;ui  ;ili  oluilii-  suliitioii  of  miai.n  onic  ;iriil  iiistcuil  of 
an  alcDliolic  s<j|ulion  of  guaiac  resin. 

*  Alkaline  solutions  should  he  m;iflc  slij^htly  arid  wilh  aictii:  acid,  as  tlic  i)luc  cnfl- 
reaction  is  very  sensitive  to  alkali. 

'' .'Kscarclli:     II  pidiclin  scz.  prat.,  lyoy. 

*  Walter:     DeiU.  med.  Woch.,  j6,  p.  309. 


BLOOD.  193 

chloride^  and  carefully  evaporate  to  dryness  over  a  low  Jianie.  Put 
a  cover  glass  in  place,  run  underneath  it  a  drop  of  glacial  acetic  acid 
and  warm  gently  until  the  formation  of  gas  bubbles  is  noted.  Add 
another  drop  of  glacial  acetic  acid,  cool  the  preparation,  examine  under 
the  microscope  and  compare  the  crystals  with  those  shown  in  Figs. 
58  and  59,  page  194.  The  haemin  crystals  result  from  the  decomposi- 
tion of  the  haemoglobin  of  the  blood.  What  are  the  steps  involved  in 
this  process  ?  The  haemin  crystals  are  also  called  Teichmann's  crystals. 
Is  this  an  absolute  test  for  blood  ?  Is  it  possible  to  differentiate  between 
human  blood  and  the  blood  of  other  species  by  means  of  the  haemin 
test? 

{b)  Atkinson  and  KendalVs  Method. — Introduce  a  small  amount 
of  the  solution  under  examination  into  a  tube  closed  at  one  end, 
add  sodium  chloride  and  glacial  acetic  acid  as  in  Teichmann's  method,^ 
fuse  or  tightly  plug  the  open  end  of  the  tube  and  heat  for  fifteen  minutes 
in  a  boiling  water-bath.^  Remove  the  tube  and  permit  it  to  cool  to 
room  temperature  spontaneously.  When  the  tube  has  cooled,  break 
it  open,  transfer  the  contents  to  a  watch  glass  or  small  evaporating 
dish  and  concentrate  on  a  water-bath  until  the  volume  of  the  fluid  in 
the  watch  glass  or  dish  has  been  reduced  to  a  few  drops.  Transfer 
a  drop  of  this  fluid  to  a  slide,  cover  with  a  cover  slip,  allow  the  slide  to 
stand  for  a  few  minutes  and  examine  it  under  a  microscope.  Com- 
pare the  crystals  with  those  shown  in  Figs.  58  and  59,  page  194.  In 
case  crystals  of  sodium  chloride  (see  Fig.  60,  page  196)  obstruct  the 
view  of  the  haemin  crystals,  dissolve  the  sodium  chloride  crystals  by 
running  a  drop  of  water  under  the  cover  slip. 

(c)  V.  Zeynek  and  Nencki's  Method. — To  10  c.c.  of  defibrinated 
blood  add  acetone  until  no  more  precipitate  forms.  Filter  off  the 
precipitated  protein  and  extract  it  with  10  c.c.  of  acetone  made  acid 
with  2-3  drops  of  hydrochloric  acid.  Place  a  drop  of  the  resulting 
colored  extract  on  a  slide,  immediately  place  a  coverglass  in  position 
and  examine  under  the  microscope.  Upon  the  evaporation  of  the 
acetone,  crystals  of  haemin  will  form.  Larger  crystals  may  be  obtained 
by  evaporating  the  acetone  extract  about  one-half,  transferring  it  to  a 
stoppered  vessel  and  allowing  it  to  remain  overnight. 

(d)  Schalfijew^ s  Method. — Place  20  c.c.  of  glacial  acetic  acid  in  a 
small  beaker  and  heat  to  80°  C.  Add  5  c.c.  of  strained  defibrinated 
blood,  again  bring  the  temperature  to  80°  C,  remove  the  flame  and 

'  Buckmaster  considers  the  use  of  potassium  chloride  preferable. 

-  Care  should  be  taken  not  to  add  too  great  an  excess  of  these  reagents. 

'  This  process  insures  constancy  of  temperature  and  strength  of  reagents. 

13 


194 


PHYSIOLOGICAL   CHEMISTRY. 


^<^  l^^v^  -^5^,'!^ 

Fig.  58. — H^MiN  Crystals  from  Human  Blood. 

Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the 

University  of  Pennsylvania. 


^^/ 


Fig.  59. — HiEMTN  Crystals  from  Siikkp  Hmjod. 

Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Rcii  licit,  of  the 

University  of  Pennsylvania. 


BLOOD.  195 

allow  the  mixture  to  cool.     Examine  the  crystals  under  the  microscope 
and  compare  them  with  those  reproduced  in  Figs.  58  and  59,  page  194. 

15.  Catalytic  Action. — To  about  10  drops  of  blood  in  a  test- 
tube  add  twice  the  volume  of  hydrogen  peroxide,  without  shaking. 
The  mixture  foams.     What  is  the  cause  of  this  phenomenon  ? 

16.  Preparation  of  Haematin. — Place  100  c.c.  of  laked  blood 
in  a  beaker  and  add  95  per  cent  alcohol  until  precipitation  ceases. 
What  bodies  are  precipitated?  Transfer  the  precipitate  to  a  flask 
and  boil  with  95  per  cent  alcohol  previously  acidulated  with  sul- 
phuric acid.  Through  the  action  of  the  acid  the  haemoglobin  is 
split  into  haematin  and  a  protein  body  called  globin.  Later  the 
"sulphuric  acid  ester  of  haematin"  is  formed,  which  is  soluble  in  the 
alcohol.  Continue  heating  until  the  precipitate  is  no  longer  colored, 
then  filter.  Partly  saturate  the  filtrate  with  sodium  chloride  and 
warm.  In  this  process  the  "hydrochloric  acid  ester  of  haematin"  is 
formed.  Filter  and  dissolve  on  the  filter  paper  by  sodium  carbonate. 
Save  this  alkaline  solution  of  haematin  and  make  a  spectroscopic  exami- 
nation later  after  becoming  familiar  with  the  use  of  the  spectroscope. 
How  does  the  spectrum  of  oxyhaemoglobin  differ  from  that  of  the 
derived  alkali  hamatin? 

17.  Variation  in  Size  of  Erythrocytes.— Prepare  two  small 
funnels  with  filter  papers  such  as  are  used  in  quantitative  analysis. 
Moisten  each  paper  with  normal  (isotonic)  salt  solution.  Into  one 
funnel  introduce  a  small  amount  of  defibrinated  ox  blood  and  into 
the  other  funnel  allow  blood  to  drop  directly  from  a  decapitated 
frog.  Note  that  the  filtrate  from  the  ox  blood  is  colored  whereas  that 
from  the  frog  blood  is  colorless.  What  deduction  do  you  make 
regarding  the  relative  size  of  the  erythrocytes  in  ox  and  frog  blood? 
Does  either  filtrate  clot?     Why? 

II.  Blood  Serum. 

1.  Coagulation  Temperature.— Place  5  c.c.  of  undiluted  serum 
in  a  test-tube  and  determine  its  temperature  of  coagulation  accord- 
ing to  the  method  described  on  page  98.  Note  the  temperature  at 
which  a  cloudiness  occurs  as  well  as  the  temperature  at  which  coag- 
ulation is  complete. 

2.  Precipitation  by  Alcohol. — To  5  c.c.  of  serum  in  a  test-tube 
add  twice  the  amount  of  95  per  cent  alcohol  and  thoroughly  mix 
by  shaking.  What  is  this  precipitate?  Make  a  confirmatory  test. 
Test  the  alcoholic  filtrate  for  protein.     Explain  the  result. 


196 


PHYSIOLOGICAL   CHEMISTRY. 


3.  Proteins  of  Blood  Seriun. — Place  about  20  c.c.  of  undiluted 
serum  in  a  small  evaporating  dish,  heat  to  boiling,  and  at  the  boiling- 
point  acidify  slightly  with  dilute  acetic  acid.  Of  what  does  this 
coagulum  consist?  Filter  off  the  coagulum  (reserve  the  filtrate)  and 
test  it  as  follows: 

(a)  Millon's  Reactian. — Make  the  test  according  to  directions 
given  on  page  ?>^. 

[h)  Hopkins-Cole  Reaction. — ]Make  the  test  according  to  direc- 
tions given  on  page  89. 


M^ 


Fig.  60. — Sodium  Chloride. 

4-  Sugar  in  Serum.— Test  a  little  of  the  filtrate  from  Experi- 
ment 3  by  Fehling's  test.     What  do  you  conclude? 

5.  Detection  of  Sodium  Chloride.— (a)  Test  a  little  of  the  filtrate 
from  Experiment  3  for  chlorides,  by  the  use  of  nitric  acid  and  argentic 
nitrate,  (b)  Evaporate  5  c.c.  of  the  filtrate  from  Experiment  3  in 
a  watch  glass  on  a  water-bath.  Plxamine  the  crystals  and  compare 
them  with  those  reproduced  in  Fig.  60,  above. 

6.  Separation  of  Serum  Globulin  and  Serum  Albumin.  Place 
10  c.c.  ol  blood  serum  in  a  small  beaker  and  saturate  with  magne- 
sium sulphate.  What  is  this  precipitate?  Filter  it  olT  and  acidify 
the  filtrate  slightly  with  acetic  acid.  What  is  this  second  precipi- 
tate? Filter  this  precipitate  off  and  test  the  filtrate  by  the  biuret 
test.     What  do  you  conclude? 

III.  Blood  Plasma. 

7.  Preparation  of  Oxalated  Plasma.  Allow  arterial  blood  to 
run  into  an  equal  volume  of  0.2  per  cent  ammonium  oxalate  solution. 


BLOOD.  197 

2.  Preparation  of  Fibrinogen. — To  25  c.c.  of  oxalated  plasma 
add  an  equal  volume  of  saturated  sodium  chloride  solution.  Note 
the  precipitation  of  fibrinogen.  Filter  off  the  precipitate  (reserve 
the  filtrate)  and  test  it  by  a  protein  color  test  (see  page  88). 

3.  Effect  of  Calcium  Salts. — Place  a  small  amount  of  oxalated 
plasma  in  a  test-tube  and  add  a  few  drops  of  a  2  per  cent  calcium 
chloride  solution.     What  occurs  ?     Explain  it. 

4.  Preparation  of  Salted  Plasma. — Allow  arterial  blood  to  run 
into  an  equal  volume  of  a  saturated  solution  of  sodium  sulphate  or  a 

JO  per  cent  solution   of  sodium  chloride.     Keep  the  mixture  in  a 
cold  place  for  about  twenty-four  hours. 

5.  Effect  of  Dilution. — Place  a  few  drops  of  salted  plasma  in  a 
test-tube  and  dilute  it  with  10-15  volumes  of  water.  What  do  you 
observe  ?     Explain  it. 

6.  Crystallization  of  Oxyhaemoglobin. — Reicherfs  Method. — 
Add  to  5  c.c.  of  the  blood  of  the  dog,  horse,  guinea-pig,  or  rat,  before 
or  after  laking,  or  defibrinating,  from  i  to  5  per  cent  of  ammonium 
oxalate  in  substance.  Place  a  drop  of  this  oxalated  blood  on  a  slide 
and  examine  under  the  microscope.  The  crystals  of  oxyhasmoglobin 
will  be  seen  to  form  at  once  near  the  margin  of  the  drop,  and  in  a  few 
minutes  the  entire  drop  may  be  a  solid  mass  of  crystals.  Compare 
the  crystals  with  those  shown  in  Figs.  50  to  56,  pages  182  to  185. 

IV.  Fibrin. 

1.  Preparation  of  Fibrin. — Allow  blood  to  flow  directly  from 
the  animal  into  a  vessel  and  rapidly  whip  it  by  means  of  a  bundle  of 
twigs,  a  mass  of  strong  cords,  or  a  specially  constructed  beater.  If 
a  pure  fibrin  is  desired  it  is  not  best  to  attempt  to  manipulate  a  large 
volume  of  blood  at  one  time.  After  the  fibrin  has  been  collected 
it  should  be  freed  from  any  adhering  blood  clots  and  washed  in  water 
to  remove  further  traces  of  blood.  The  pure  product  should  be  very 
light  in  color.  It  may  be  preserved  under  glycerol,  dilute  alcohol,  or 
chloroform  water. 

2.  Solubility. — Try  the  solubility  of  small  shreds  of  freshly 
prepared  fibrin  in  the  usual  solvents. 

3.  Millon's  Reaction. — Make  the  test  according  to  directions 
given  on  page  88. 

4.  Hopkins-Cole  Reaction. — Make  the  test  according  to  direc- 
tions given  on  page  89. 

5.  Biuret  Test. — Make  the  test  according  to  directions  given  on 
page  90. 


iqS  physiological  chemistry. 

V.  Detection  of  Blood  in  Stains  on  Cloth,  etc. 

1.  Identification  of  Corpuscles. — If  the  stain  under  examina- 
tion is  on  cloth  a  portion  should  be  extracted  with  a  few  drops  of 
glycerol  or  normal  (0.9  per  cent)  sodium  chloride  solution.  A  drop 
of  this  solution  should  then  be  examined  under  the  microscope  to 
determine  if  corpuscles  are  present. 

2.  Tests  on  Aqueous  Extract. — A  second  portion  of  the  stain 
should  be  extracted  with  a  small  amount  of  water  and  the  following 
tests  made  upon  the  aqueous  extract: 

(a)  Hcemochromogen. — Make  a  small  amount  of  the  extract  al- 
kaline by  potassium  hydroxide  or  sodium  hydroxide,  and  heat  until 
a  brownish-green  color  results.  Cool  and  add  a  few  drops  of  ammo- 
nium sulphide  or  Stokes'  reagent  (see  page  199)  and  make  a  spectro- 
scopic examination.  Compare  the  spectrum  with  that  of  hsemo- 
chromogen  (see  Absorption  Spectra,  Plate  II). 

(6)  Hdmin  Test. — Make  this  test  upon  a  small  drop  of  the  aque- 
ous extract  according  to  the  directions  given  on  page  192. 

(c)  Guaiac  T^^/.— Make  this  test  on  the  aqueous  extract  accord- 
ing to  the  directions  given  on  page  191.  The  guaiac  solution  may 
also  be  applied  directly  to  the  stain  without  previous  extraction  in 
the  following  manner:  Moisten  the  stain  with  water,  and  after  allow- 
ing it  to  stand  several  minutes,  add  an  alcoholic  solution  of  guaiac 
(strength  about  i  :6o)  and  a  little  hydrogen  peroxide  or  old  turpentine. 
The  customary  blue  color  will  be  observed  in  the  presence  of  blood. 

{d)  Benzidine  Reaction. — Make  this  test  according  to  directions 
given  on  p.  192. 

(e)  Acid  Hcematin. — If  the  stain  fails  to  dissolve  in  water  extract 
with  acid  alcohol  and  examine  the  spectrum  for  absorption  bands 
of  acid  ha^matin  (see  Absorption  Spectra,  Plate  II). 

VI.  Spectroscopic  Examination  of  Blood. 

(For  Absorption  Spectra  sec  Plates  I.  and  11.) 

Either  the  angular-vhion  spectroscope  (Figs.  62  and  63,  page  200) 
or  the  direct-\\'r\(m  spectroscope  (Fig.  61,  ])ugc  199)  may  be  used  in 
making  the  spectroscopic  examination  of  the  blood.  For  a  com- 
plete description  of  these  instruments  the  student  is  referred  to  any 
standard  text-book  of  physics. 

I.  Oxyhaemoglobin. — Examine  dilute  (1:50)  defibrinalcd  blood 
spectrosco])icully.  No|e  the  broad  absorption-band  between  D  and 
\-\.     ('ontinue  the  dilution  until  this  single  broad  band  gi\es  ])lace  to 


BLOOD. 


199 


two  narrow  bands,  the  one  nearer  the  D  line  being  the  narrower. 
These  are  the  typical  absorption-bands  of  oxyhgemoglobin  obtained 
from  dilute  solutions  of  blood.  Now  dilute  the  blood  very  freely  and 
note  that  the  bands  gradually  become  more  narrow  and,  if  the  dilution 
is  sufficiently  great,  they  finally  entirely  disappear. 

2.  Haemoglobin  (so-called  Reduced  Haemoglobin). — To  blood 
which  has  been  diluted  sufficiently  to  show  well  defined  oxyhsemo- 
globin  absorption-bands  add  a  small  amount  of  Stokes'  reagent.^ 
The  blood  immediately  changes  in  color  from  a  bright  red  to  violet- 


FiG.  61. — Direct- VISION  Spectroscope. 


red.  The  oxyhaemoglobin  has  been  reduced  through  the  action  of 
Stokes'  reagent  and  haemoglobin  (so-called  reduced  haemoglobin) 
has  been  formed.  This  has  been  brought  about  by  the  removal 
of  some  of  the  loosely  combined  oxygen  from  the  oxyhaemoglobin. 
Examine  this  haemoglobin  spectroscopically.  Note  that  in  place  of 
the  two  absorption  bands  of  oxyhaemoglobin  we  now  have  a  single 
broad  band  lying  almost  entirely  between  D  and  E.  This  is  the 
typical  spectrum  of  haemoglobin.  If  the  solution  showing  this  spectrum 
be  shaken  in  the  air  for  a  few  moments  it  will  again  assume  the  bright 
red  color  of  oxyhaemoglobin  and  show  the  characteristic  spectrum  of 
that  pigment. 

3.  Carbon  Monoxide  Haemoglobin. — The  preparation  of  this 
pigment  may  be  easily  accomplished  by  passing  ordii^ary  illuminating 
gas^  through  defibrinated  ox-blood.  Blood  thus  treated  assumes  a 
brighter  tint  (carmine)  than  that  imparted  by  oxyhaemoglobin.  In 
very  dilute  solution  oxyhaemoglobin  appears  yellowish-red  whereas 
carbon  monoxide  haemoglobin  under  the  same  conditions  appears 
bluish-red.  Examine  the  carbon  monoxide  haemoglobin  solution 
spectroscopically.  Observe  that  the  spectrum  of  this  body  resembles 
the   spectrum   of   oxyhaemoglobin   in   showing  two   absorption-bands 

'  Stokes'  reagent  is  a  solution  containing  2  per  -cent  ferrous  sulphate  and  3  per  cent 
tartaric  acid.  When  needed  for  use  a  small  amount  should  be  placed  in  a  test-tube  and 
ammonium  hydroxide  added  until  the  precipitate  which  forms  on  the  first  addition  of  the 
hydroxide  has  entirely  dissolved.  This  produces  ammonium  ferrotartrate  which  is  a 
reducing  agent. 

-  The  so-called  water  gas  with  which  ordinary  illuminating  gas  is  diluted  contains 
usually  as  much  as  20  per  cent  of  carbon  monoxide  (CO). 


200 


PHYSIOLOGICAL    CHEMISTRY. 


between  D  and  E.  The  bands  of  carbon  monoxide  haemoglobin,  how- 
ever, are  somewhat  nearer  the  violet  end  of  the  spectrum.  Add  some 
Stokes'  reagent  to  the  solution  and  again  examine  spectroscopically. 


Fig.  62. — Angul.'\r-visiox  Spectroscope  Arranged  for  Absorptiox  .Analysis. 

Note  that  the  position  and  intensity  of   the  absorption-bands  remain 
unaltered. 

The  following  is  a  delicate  chemical  test  for  the  detection  of  carbon 
monoxide  haemoglobin : 


Fig.  05.— Jji.xGKA.vi  ov  Angular-vision  Spectkoscui-k.  (f^ong.) 
The  white  light  F  enters  the  collimator  tu])C  through  a  narrow  slit  an<l  jiasses  to  tlie 
prism,  P,  which  has  the  jjower  of  refracting  and  dispersing  the  light.  'I'he  rays  then  i)ass 
to  the  double  convex  lens  of  the  oi  ular  tulie  and  are  fle(le(  ted  to  the  eye-i)iece  E.  The 
flotled  lines  show  the  magnified  virtual  image  which  is  ff)rmed.  The  third  tube  contains  a 
scale  whose  image  is  relleded  into  the  ocular  and  shown  with  the  spectrum.  Between  the 
light  /'■  and  the  collimator  slit  is  placed  a  cell  to  hold  the  solution  undergoing  examination. 

Tannin  J'e.sl.  l)\\U\c  the  Ijhjod  to  be  tested  into  two  portions  and 
dilute  each  with  four  volumes  of  distilled  water.  Place  the  diluted 
bloofj   mixtures   in   two  siu.ill    iLisl.--   or   l;irge   test  tubes  and   add    20 


BLOOD.  20I 

drops  of  a  lo  per  cent  solution  of  potassium  ferricyanide.^  Allow 
both  solutions  to  stand  for  a  few  minutes,  then  stopper  the  ^■essels  and 
shake  one  vigorously  for  10-15  minutes,  occasionally  removing  the 
stopper  to  permit  air  to  enter  the  vessel.^  Add  5-10  drops  of  am- 
monium sulphide  (yellow)  and  10  c.c.  of  a  10  per  cent  solution  of  tannin 
to  each  flask.  The  contents  of  the  shaken  flask  will  soon  exhibit  the 
formation  of  a  dirty  olive  green  precipitate,  whereas  the  flask  which 
was  not  shaken  and  which,  therefore,  still  contains  carbon  monoxide 
haemoglobin,  will  exhibit  a  bright  red  precipitate,  characteristic  of 
carbon  monoxide  haemoglobin.  This  test  is  more  delicate  than  the 
spectroscopic  test  and  serves  to  detect  the  presence  of  as  low  a  content 
as  5  per  cent  of  carbon  monoxide  haemoglobin. 

4.  Neutral  Methaemoglobin. — Dilute  a  Httle  defibrinated  blood 
(i  :  10)  and  add  a  few  drops  of  a  freshly  prepared  10  per  cent  solution  of 
potassium  ferricyanide.  Shake  this  mixture  and  observe  that  the 
bright  red  color  of  the  blood  is  displaced  by  a  brownish  red.  Now  dilute 
a  little  of  this  solution  and  examine  it  spectroscopically.  Note  the 
single,  very  dark  absorption-band  lying  to  the  left  of  D,  and,  if  the 
dilution  is  sufficiently  great,  also  observe  the  two  rather  faint  bands 
lying  between  D  and  E  in  somewhat  similar  positions  to  those  occupied 
by  the  absorption  bands  of  oxyhaemoglobin.  Add  a  few  drops  of 
Stokes'  reagent  to  the  methaemoglobin  solution  while  it  is  in  position 
before  the  spectroscope  and  note  the  immediate  appearance  of  the 
oxyhaemoglobin  spectrum  which  is  quickly  followed  by  that  of 
haemoglobin. 

5.  Alkaline  Methaemoglobin. — Render  a  neutral  solution  of 
methaemoglobin,  such  as  that  used  in  the  last  experiment  (4),  slightly 
alkaline  with  a  few  drops  of  ammonia.  The  solution  becomes  redder 
in  color,  due  to  the  formation  of  alkaline  methaemoglobin  and  shows 
a  spectrum  different  from  that  of  the  neutral  body.  In  this  case 
we  have  a  band  on  either  side  of  D,  the  one  nearer  the  red  end  of  the 
spectrum  being  much  the  fainter.  A  third  band,  darker  than  either 
of  those  mentioned,  lies  between  D  and  E  somewhat  nearer  E. 

6.  Alkali  Haematin. — Observe  the  spectrum  of  the  alkali  haem- 
atin  prepared  in  Experiment  16  on  page  195.  Also  malie  a  spectro- 
scopic   examination    of    a    freshly    prepared    alkali    haematin.^     The 


'  This  transforms  the  oxyhaemoglobin  into  methfemoglobin. 

-  This  is  done  to  free  the  blood  from  carbon  monoxide  haemoglobin. 

■'  Alkali  hccmatin  may  be  prepared  by  mixing  one  -v'olume  of  a  concentrated  potassium 
hydroxide  or  sodium  hydroxide  solution  and  two  volumes  of  dilute  (i:")  defibrinated  blood. 
This  mixture  should  be  heated  gradually  almost  to  boiling,  then  cooled  and  shaken  for  a 
few  moments  in  the  air  before  examination. 


202  PHYSIOLOGICAL    CHEMISTRY. 

typical  spectrum  of  alkali  hcTmatin  shows  a  single  absorption-band 
lying  across  D  and  mainly  toward  the  red  end  of  the  spectrum. 

7.  Reduced  Alkali  Haematin  or  Hsemochromogen. — Dilute 
the  alkali  haematin  solution  used  in  the  last  experiment  (6)  to  such 
an  extent  that  it  shows  no  absorption  band.  Now  add  a  few  drops 
of  Stokes'  reagent  and  note  that  the  greenish-brown  color  of  the 
alkali  haematin  solution  is  displaced  by  a  bright  red  color.  This  is 
due  to  the  formation  of  haemochromogen  or  reduced  alkali  haematin. 
Examine  this  solution  spectroscopically  and  observe  the  narrow, 
dark  absorption-band  lying  midway  between  D  and  E.  If  the  dilu- 
tion is  not  too  great  a  faint  band  may  be  observed  in  the  green  extend- 
ing across  E  and  b. 

8.  Acid  Hagmatin. — To  some  defibrinated  blood  add  half  its  vol- 
ume of  glacial  acetic  acid  and  an  equal  volume  of  ether.  Mix  thor- 
oughly. The  acidified  ethereal  solution  of  haematin  rises  to  the  top 
and  may  be  poured  off  and  used  for  the  spectroscopic  examination. 
If  desired  it  may  be  diluted  with  acidified  ether  in  the  ratio  of  one  part 
of  glacial  acetic  acid  to  two  parts  of  ether.  A  distinct  absorption- 
band  will  be  noted  in  the  red  between  C  and  D  and  lying  somewhat 
nearer  C  than  the  band  in  the  methaemoglobin  spectrum.  Between 
D  and  F  may  be  seen  a  rather  indistinct  broad  band.  Dilute  the  solu- 
tion until  this  band  resolves  itself  into  two  bands.  Of  these  the  more 
prominent  is  a  broad,  dark  absorption-band  lying  in  the  green  between 
b  and  F.  The  second,  a  narrow  band  of  faint  outline,  lies  in  the  light 
green  to  the  red  side  of  E.  A  fourth  very  faint  band  may  be  observed 
lying  on  the  violet  side  of  D. 

9.  Acid  Haematoporphyrin. — To  5  c.c.  of  concentrated  sul- 
phuric acid  in  a  test-tube  add  two  drops  of  blood,  mixing  thoroughly 
by  agitation  after  the  addition  of  each  drop.  A  wine-red  solution 
is  produced.  Examine  this  solution  spectroscopically.  Acid  haem- 
atoporphyrin gives  a  spectrum  with  an  absorpti()n-])and  on  cither 
side  of  D,  the  one  nearer  the  red  end  of  the  spectrum  Ijcing  the 
narrower. 

10.  Alkaline  Haematoporphyrin. — Introduce  the  acid  haemato- 
pory)hyrin  soluti(jn  just  examined  into  an  excess  of  distilled  water. 
Cool  the  solution  and  add  yjotassium  hydroxide  slowly  until  the 
reaction  is  but  slightly  acid.  A  colored  precipitate  forms  which 
includes  the  princiyjal  jjorlion  of  the  haematoporphyrin.  The  ])resence 
of  sodium  acetate  facilitates  the  formation  of  this  ]jrecij)itate.  Filter 
off  the  precipitate  and  dissolve  it  in  a  small  amount  of  dilute  potassium 
hydroxide.     Alkaline  haematoporphyrin  prepared  in  this  way  forms 


BLOOD. 


203 


a  bright  red  solution  and  possesses  four  absorption-bands.  The 
first  is  a  very  faint,  narrow  band  in  the  red,  midway  between  C  and  D; 
the  second  is  a  broader,  darker  band  lying  across  D,  principally  to  the 
violet  side.  The  third  absorption-band  lies  principally  between  D 
and  E,  extending  for  a  short  distance  across  E  to  the  violet  side,  and 
the  fourth  band  is  broad  and  dark  and  lies  between  b  and  F.  The 
first  band  mentioned  is  the  faintest  of  the  four  and  is  the  first  to  disap- 
pear when  the  solution  is  diluted. 


VII.  Instriunents  Used  in  the  Clinical  Examination  of  the  Blood. 

I.  Fleischl's  Haemometer  (Fig.  64,  below). — This  is  an  instru- 
ment used  quite  extensively  clinically,  for  the  quantitative  deter- 
mination of  haemoglobin.  The  instrument  consists  of  a  small  cylinder 
which  is  provided  with  a  fixed  glass  bottom  and  a  movable  glass  cover, 
and  which  is  divided,  by  means  of  a  metal  septum,  into  two  compart- 
ments of  equal  capacity.  This  cylinder  is  supported  in  a  vertical  posi- 
tion by  means  of  a  mechanism  which  resembles  the  base  and  stage  of  an 
ordinary  microscope.  Underneath  the  stage  is  placed  a  colored  glass 
wedge  (see  Fig.  66,  p.  204),  so  arranged  as  to  run  immediately  beneath 
the  glass  bottom  of  one  of  the  compartments  of  the  cylinder  and  ground 
in  such  a  manner  that  each  part  of 
the  wedge  corresponds  in  color  to  a 
solution  of  haemoglobin  of  some  defi- 
nite percentage.  The  glass  wedge  is 
held  in  a  metal  frame  and  may  be 
moved  backward  or  forward  by  means 
of  a  rack  and  pinion  arrangement.  A 
scale  along  the  side  of  this  frame 
indicates  the  percentage  of  the  normal 
amount  of  haemoglobin  which  each 
particular  variation  in  the  depth  of 
color  of  the  ground  wedge  represents, 
taking  the  normal  haemoglobin  content 
as  100.^  In  a  position  corresponding  to  the  position  of  the  mirror 
on  the  ordinary  microscope  is  attached  a  light-colored  opaque  plate 
which  serves  to  reflect  the  light  upward  through  the  colored  wedge  and 
the  cylinder  to  the  eye  of  the  observer. 

In  making  a  determination  of  the  percentage  of  haemoglobin  by 
this  instrument  the  procedure  is  as  follows:  Fill  each  compartment 

^  The  scale  of  the  ordinary  instrument  is  usually  too  high. 


Fig.  64. 


-Fleischl's    H-emometer. 
{Da  Costa.) 


204 


PHYSIOLOGICAL    CHEMISTRY. 


Fig.     65. —  Pipette     of 

FLEISCHL'S  H.EMOMETER. 


about  three-fourths  full  of  distilled  water.  Puncture  the  finger- 
tip or  lobe  of  the  ear  of  the  subject  by  means  of  a  sterile  needle  or 
scalpel  and,  as  soon  as  a  drop  of  blood  appears,  place  one  end  of 
the  capillary  pipette  (Fig.  65),  which  accompanies  the  instrument, 
against  the  drop  and  allow  it  to  fill  by  capillary  attraction.  To  prevent 
the  blood  from  adhering  to  the  exterior  of  the  tube,  and  so  render  the 
determination  inaccurate,  it  is  customary  to  apply  a  very  thin  coating 
of  mutton  fat  to  the  outer  surface  before  using  or 
to  wrap  the  tube  in  a  piece  of  oily  chamois  when 
not  in  use.  As  soon  as  the  tube  has  been  ac- 
curately filled  with  blood  it  should  be  dipped 
into  the  water  of  one  of  the  compartments  of  the 
cylinder  and  all  traces  of  the  blood  washed  out 
with  water  by  means  of  a  small  dropper  which 
accompanies  the  instrument.  If  the  blood  is 
not  well  distributed  throughout  the  compartment  and  does  not 
form  a  homogeneous  solution  the  contents  of  the  compartment 
should  be  mixed  thoroughly  by  means  of  the  metal  handle  of  'the 
capillary  measuring  pipette.  When  this  has  been  done  each  com- 
partment should  be  completely  filled  with  distilled  water  and  the 
glass  cover  adjusted,  care  being  taken  that  the  contents  of  the 
two  compartments  do  not  mix.  Now  adjust  the  cylinder  so  that  the 
compartment  containing  the  pure  distilled  water  is  immediately 
abo\e  the  colored  glass  wedge.  By  means  of  the  rack  and  pinion 
arrangement  manipulate  the  colored  wedge  until  a  portion  of  it  is 
found  which  corresponds  in  color  with  the  diluled  l)l()od.  When  this 
agreement  in  color  has  been  se- 
cured the  point  on  the  scale  cor 
responding  to  this  particular  color 
should  be  read  and  the  actual 
percentage  of  haemoglobin  com- 
puted. For  instance,  if  the  scale 
reading  is  90  it  means  that  the 
blood  under  examination  contains 

90  ])cr  cent  of  the  normal  (luanlil)'  of  hiemoglohin,  /.  r.,  ()0  percent 
of  14  jjcr  cent. 

2.  Fleischl-Miescher  Haemometer.  'Ihe  apparatus  of  {''leischl 
has  recently  been  modificfl  by  Miescher.  If  all  precautions  are  taken, 
the  margin  of  error  in  the  absolute  quantity  of  haemoglobin  determined 
i>y  this  instrument  does  not  exceed  o.  15  0.22  ])er  cent  by  weight  of 
the  blood.     Detailed  directions  for  the  manij>ulalion  of  the  Fleischl- 


i 


0        llO     ao      J|0     <1|0      5|0     610      7|0     BjO     010     100     no     120 

1 1  I  1 1  I  j  I  ii  I  I  r  I  1 1  1 1  I  1 1  1 1 1  fi 


Fio.    66. — Colored    Glass   Wkdck    of 

FLIOISCin.'s    II.KMOMKTKR.       (Pil  Co.slll.) 


BLOOD.  205 

Miescher  hasmometer  accompany  the  instrument.  In  brief  Miescher 
modified  the  instrument  as  follows:  (i)  The  scale  of  each  instrument 
is  supplied  with  a  caliber  table  of  absolute  haemoglobin  values,  expressed 
in  milligrams:  the  scale  of  Fleischl's  haemometer  shows  the  percentage 
of  haemoglobin  in  relation  to  an  average  selected  somewhat  arbitrarily. 
Thus  many  errors  arising  from  the  irregular  coloring  of  the  glass  wedge 
of  the  older  apparatus  are  avoided  in  the  instrument  as  modified. 
(2)  Each  instrument  is  accompanied  by  a  measuring  pipette  (melan- 
geur)  which  allows  of  a  more  accurate  measurement  of  the  blood  than 
was  possible  with  the  capillary  tubes  of  the  older  apparatus.  (3 )  With 
the  aid  of  the  measuring  pipette  mentioned  above  blood  of  varying 
degrees  of  concentration  may  be  compared.  In  this  way  the  in- 
dividual examinations  are  controlled  and  a  check  upon  the  accuracy 
of  the  graduation  in  the  color  of  the  glass  wedge  is  also  afforded.  This 
wedge  is  much  more  evenly  and  accurately  colored  than  in  the  un- 
modified apparatus  of  Fleischl.  (4)  Before  reading  the  percentage 
as  indicated  by  the  scale,  the  chamber  is  covered  with  a  glass  and  a 
diaphragm  which  sharply  define  the  field  on  all  sides  without  the 
formation  of  a  meniscus. 

The  measuring  pipette  is  constructed  essentially  the  same  as  the 
pipettes  which  accompany  the  Thoma-Zeiss  apparatus  (see  page  209). 
The  capillary  portion,  however,  is  graduated,  i,  2/3  and  i/  2  which 
enables  the  observer  to  dilute  the  blood  sample  in  the  proportion 
of  1:200,  1:300  or  1:400  as  he  may  desire.  If  there  is  difficulty  in 
drawing  in  the  blood  exactly  to  one  of  the  graduations  just  mentioned 
the  amount  of  blood  above  or  below  the  volume  indicated  by  the  grad- 
uation may  be  determined  by  means  of  certain  delicate  cross-lines  which 
are  placed  directly  above  and  below  the  graduation.  Each  cross-line 
corresponds  to  i/ioo  of  the  volume  of  the  capillary  tube  from  the  tip 
to  the  I  graduation. 

A  o.  I  per  cent  solution  of  sodium  carbonate  is  used  to  dissohe  the 
stroma  of  the  erythrocytes  and  so  render  the  blood  solution  perfectly 
clear.  If  this  is  not  done  the  color  of  the  blood  solution  invariably 
appears  darker  in  tone  than  that  of  the  colored  glass  wedge.  A  freshly 
prepared  sodium  carbonate  solution  should  be  used  in  order  that  the 
clearness  of  the  solution  may  not  be  marred  by  the  presence  of  sodium 
bicarbonate. 

3.  Dare's  Haemoglobinometer  (Fig.  67). — This  instrument,  as 
the  name  signifies,  is  used  for  the  determination  of  haemoglobin.  In 
using  either  Fleischl's  haemometer  or  the  instrument  as  modified  by 
Miescher  the  blood  is  diluted  for  examination,  whereas  with  the  Dare 


2o6 


PHYSIOLOGICAL   CHEMISTRY. 


instrument  no  diliitian  is  required.  This  probably  allows  of  rather 
more  accurate  determinations  than  are  possible  with  the  old  Fleischl 
apparatus. 

The  instrument  consists  essentially  of  the  following  parts:  (i) 
A  capillary  observation  cell,  {2)  a  semicircular  colored  glass  wedge, 
(3)  a  milled  wheel  for  manipulating  the  wedge,  (4)  a  candle  used 
to  illuminate  portions  of  the  capillary  observation  cell  and  the  colored 

wedge,  (5)  a  small  telescope 
used  in  the  examination  of  the 
areas  illuminated  by  the  candle 
flame,  (6)  a  scale  graduated  in 
percentages  of  the  normal 
amount  of  haemoglobin,  (7)  a 
hard-rubber  case,  (8)  a  mova- 
ble screen  attached  to  the  case. 
The  capillary  observation 
cell  is  formed  of  two  small, 
polished  rectangular  plates  of 
glass,  one  being  transparent 
and  the  other  opaque.  When 
held  in  position  on  the  instru- 
ment, by  means  of  a  small 
metal  bracket,  the  opaque  por- 
tion of  the  cell  is  nearer  the 
candle  and  thus  serves  to  soften 
the  glare  of  light  when  an  ob- 
servation is  being  made.  The 
transparent  portion  of  the  cell 
is  directly  over  a  circular  open- 
ing in  the  case,  through  which 
the  blood  specimen  is  viewed 
by  means  of  the  small  telescope. 
The  semicircular  colored  glass  wedge  is  so  ground  that  each  par- 
ticular shade  of  color  corresponds  to  that  possessed  by  fresh  blood 
which  contains  some  definite  percentage  of  haemoglobin.  It  is  mounted 
upon  a  disc  which  may  be  manipulated  by  the  milled  wheel  in  such  a 
manner  as  to  bring  successive  portions  of  the  wedge  in  ])()sition  to  be 
viewed  through  a  circular  opening  contiguous  to  the  (Opening  through 
which  the  blood  specimen  is  viewed.  For  a  further  description  of  the 
instrument  see  Figs.  67,  68,  and  69. 

In  using  the  Dare  ha^moglobinometcr  i>rocecd  as  follows:  Puncture 


Fig.  67. — Dare's  H.5;moglobinometer.  {Da 
Costa.) 
R,  Milled  wheel  acting  by  a  friction  bearing 
on  the  rim  of  the  color  disc;  S,  case  inclosing 
color  disc,  and  provided  with  a  stage  to  which 
the  blood  chamber  is  fitted;  T,  movable  wing 
which  is  swung  outward  during  the  observation, 
to  serve  as  a  screen  for  the  observer's  eyes,  and 
which  acts  as  a  cover  to  inclose  the  color  disc 
when  the  instrument  is  not  in  use;  U,  telescop- 
ing camera  tube,  in  position  for  examination; 
V,  aperture  admitting  light  for  illumination  of 
the  color  disc;  X,  capillary  blood  chamber 
adjusted  to  stage  of  instrument,  the  slip  of 
opafjue  glass,  W,  being  nearest  to  the  source  of 
light;  Y,  detachable  candle-holder;  Z,  rect- 
angular slot  through  which  the  ha;mogloVjin 
scale  indicated  on  the  rim  of  the  color  disc  is 
read. 


BLOOD. 


20: 


the  finger-tip  or  lobe  of  the  ear  of  the  subject  by  means  of  a  needle  or 
scalpel  and,  after  a  drop  of  blood  of  good  proportions  has  formed, 
place  the  fiat  capillary  observation  cell  in  contact  with  the  drop  and 
allow  it  to  fill  by  capillary  attraction  (Fig.  69).  Replace  the  cell  in 
its  proper  place  on  the  instrument.  When 
in  position,  a  portion  of  this  cell  may  be 
observed  through  a  small  telescope  attached 
to  the  apparatus.  It  is  viewed  through  a 
circular  opening  and  near  this  circle  is  a 
second  one  through  which  a  portion  of  a 
semicircular  colored  glass  wedge  is  visible. 
These  two  circles  are  illuminated  simul- 
taneously by  means  of  the  flame  of  a 
candle.  The  colored  glass  may  be  rotated 
by  means  of  a  milled  wheel  and  the  point 
of  agreement  of  the  color  of  the  adjoining 
discs  may  be  determined  in  the  same  way 
as  in  Fleischl's  haemometer.  The  scale 
reading  gives  the  percentage  of  the  normal 
quantity  of  haemoglobin  which  the  blood  sample  under  examination 
contains.  Compute  the  actual  haemoglobin  content  in  the  same  manner 
as  from  the  scale  reading  of  the  Fleischl  haemometer  (see  page  204). 

4.  Tallquist's  Haemoglobin  Scale. — This  consists  essentially  of 
a  series  of  ten  colors  corresponding  to  stains  produced  by  blood  con- 
taining varying  percentages  of  haemoglobin.     In  using  this  scale  a  drop 


Fig.  68. — Horizontal  Sec- 
tion OF  Dare's  H^moglo- 
BiNOMETER.     (Da  Costa.) 


Fig.  69. — Method  of  Filling  the  Capillary  Observation  Cell  of  Dare's  H.emo 

GLOBINOMETER.       {Da  Costa.) 


of  blood  is  allowed  to  fall  on  a  small  section  of  filter  paper  and  the 
resulting  color  is  compared  with  the  ten  colors  of  the  scale.  When  the 
color  in  the  scale  is  found  which  corresponds  to  the  color  of  the  blood 
stain  the  accompanying  haemoglobin  value  is  read  off  directly.     This 


208 


PHYSIOLOGICAL    CHEMISTRY. 


is  a  ven'  convenient  method  for  determining  haemoglobin  at  the  bedside. 
There  is  a  possibility  of  the  colors  being  inaccurately  printed,  however, 
and  even  if  originally  correct  in  tint,  under  the  continued  influence  of 
air  and  light  they  must  e^"entually  alter  somewhat. 

5.  Thoma-Zeiss  Haemocytometer. — This  is  an  instrument  used 
in  "blood  counting,"  i.  f.,  in  determining  the  number  of  erythrocytes 
and  leucocytes.  The  instrument  consists  of  a  microscopic  slide 
constructed  of  heaxy  glass  and  provided  with  a  central  counting  cell 
(see  Fig.  70,  below).  This  cell,  with  the  coverglass  in  position,  is 
exactly  o.i  millimeter  deep.  The  floor  of  the  cell  is  divided  by 
delicate  lines  into  squares  each  of  which  is  i  400  of  a  square  millimeter 
in  area  (see  Fig.  72,  page  210).  The  volume  of  blood  therefore  between 
any  particular  square  and  the  coverglass  above  must  be  i  4000  culjic 
millimeter.     Accompanying  each  instrument  are  two  capillary  pipettes 


Fig.  70. — Thu-M.a-Zeiss  Counting  Chamber.     {Da  Costa.) 


(Fig.  71,  page  209),  each  constructed  with  a  mixing  bull.i  in  its  upper 
portion.  Each  bulb  is  further  provided  with  an  enclosed  glass  bead 
which  is  of  great  assistance  in  mixing  the  contents  of  the  chamber.  The 
stem  of  each  pipette  is  graduated  in  tenths  from  the  tip  to  the  bulb. 
The  final  graduation  at  the  upper  end  of  the  bulb  is  10 1  on  the  pipette 
used  in  mixing  the  blood  sample  in  which  the  erythrocytes  arc  counted 
(erythrocytomeler,  see  Fig.  71,  page  209),  and  1 1  on  the  pipette  used  in 
mixing  the  blood  sample  for  the  leucocyte  count  (leucocytometer,  see 
Fig.  71,  page  209;.  In  making  "blood  counts"  with  the  haemo- 
cytometer it  is  necessary  to  use  some  diluting  fluid.  Two  very  satis- 
factory fcjrms  of  fluid  for  this  })urpose  are  'I'oison's  and  Sherrington's 
solutions.*     When  either  of  these  solutions  is  used  as  the  dikiling  tluid 

'  Toison's    solution    has  tho    following         Slu-rrington's  solution   has  the  following 

formula:  formula; 

Methyl-violet  .  .  0.025  gram.  Mt'hylene-itluc o.i  gram. 

SofJium  chUmdf.  i  gram.  Sodium  chloride 1.2  gram. 

Sodium  sulphate 3  grams.  Neutral  jjotassiuni  oxalate.  .    .      12  gram. 

(ilyierr^l ?o  grams.  Distilled  water ,iOO.o  grams. 

Distilled  water 160  grams. 


BLOOD. 


209 


it  is  possible  to  make  a  very  satisfactory  count  of  both  the  erythrocytes 
and  leucocytes  from  the  same  preparation,  since  the  leucocytes  are 
stained  by  the  methyl-violet  or  methylene-blue. 

In  counting  the  erythrocytes  by  means  of  the  hsemocytometer, 
proceed  as  follows:  Thoroughly  cleanse  the  tip  of  the  finger  or  lobe  of 
the  ear  of  the  subject  by  the  use  of  soap  and  water, 
alcohol  and  ether  applied  in  the  sequence  just  given. 
Puncture  the  skin  by  means  of  a  needle  or  scalpel 
and  allow  the  blood  drop  to  form  without  pressure. 
Place  the  tip  of  the  pipette  in  contact  with  the  blood 
drop,  being  careful  to  avoid  touching  the  skin,  and 
draw  blood  into  the  pipette  up  to  the  point  marked 
0.5  or  I  according  to  the  desired  dilution.  Rapidly 
wipe  the  tip  of  the  pipette  and  immediately  fill  it  to 
the  point  marked  loi  with  Toison's  or  Sherring- 
ton's solution.  Now  thoroughly  mix  the  blood  and 
diluting  fluid  within  the  mixing  chamber  by  tap- 
ping the  pipette  gently  against  the  finger,  or  by 
shaking  it  while  held  securely  with  the  thumb  at 
one  end  and  the  middle  finger  at  the  other.  After 
the  two  fluids  have  been  thoroughly  mixed  the 
diluting  fluid  contained  in  the  capillary-tube  below 
the  bulb  should  be  discarded  in  order  to  insure  the 
collection  of  a  drop  of  the  thoroughly  mixed  blood 
and  diluting  solution  for  examination.  Transfer  a 
drop  from  the  pipette  to  the  ruled  floor  of  the 
counting  chamber  and,  after  placing  the  cover- 
glass  firmly  in  position,^  allow  an  interval  of  a  few 
minutes  to  elapse  for  the  corpuscles  to  settle  before 
making  the  count.  Now  place  the  slide  under  the 
microscope  and  count  the  number  of  erythrocytes 
in  a  number  of  squares,  counting  the  corpuscles 
which  are  in  contact  with  the  upper  and  the  right-hand  boundaries  of 
the  square  as  belonging  to  that  square.  Take  the  squares  in  some 
definite  sequence  in  order  that  the  recounting  of  the  same  corpuscles 
may  be  avoided.  Of  course,  all  things  being  equal,  the  greater  the 
number  of  squares  examined  the  more  accurate  the  count.  It  is 
considered  essential  under  all  circumstances,  where  an  accurate  count 
is  desired,  that  the  counting  chamber  shall  be  filled,  at  least  twice,  and 


B 

Fig.  71. — Thoma- 
Zeiss        Capillary 
Pipettes. 

A,  Erythrocytometer; 

B,  Leucocytometer. 


^  If  the  cuverglass  is  in  accurate  apposition  to  the  counting  cell  Newton's  rings  may  be 
plainly  observed. 

14 


2IO  PHYSIOLOGICAL    CHEMISTRY. 

the    individual    counts    made   in   each  instance,  as  indicated  above, 
before  the  data  are  deemed  satisfactory. 

To  calculate  the  number  of  erythrocytes  per  cubic  millimeter  of 
undiluted  blood  proceed  as  follows:  Determine  the  number  of  cor- 
puscles in  any  given  number  of  squares  and  divide  this  total  by  the 
number  of  squares,  thus  obtaining  the  average  number  of  erythrocytes 
per  square.     Multiply  this  average  by  4,000  to  obtain  the  number 


Fig.  72. — Ordinary  Ruling  of  Thoma-Zejss  Counting  Cbdxmber.     {Da  Costa.) 

of  erythrocytes  per  cubic  millimeter  of  diluted  blood,  and  multiply 
this  product  by  jog  or  200,  according  to  the  dilution,  to  obtain  the 
number  of  erythrocytes  per  cubic  millimeter  of  undiluted  blood.     Thus: 

Average   number  of  erythrocytes  Number  of  erythrocytes   per 

per  square  ^  ^  cubic  millimeter. 

Great  care  should  be  taken  to  see  that  the  capillary  pipette  is  prop- 
erly cleaned.  After  using,  it  should  be  immediately  rinsed  out  with 
the  diluting  fluid,  then  with  water,  alcohol,  and  ether  in  the  sequence 
given.  Finally  dry  air  should  be  drawn  through  the  capillary  and  a 
horse  hair  inserted  to  prevent  the  entrance  of  dust  particles. 

In  counting  leucocytes  by  means  of  the  haemocytomcter  proceed 
as  follows:  As  menlioncfl  above,  if  the  diluting  fluid  is  either  Toison's 
or  Sherrington's  solution  the  leucocytes  may  be  counted  in  the  same 
specimen  of  blood  in  which  the  erythrocytes  are  counted.  When 
this  is  done  it  is  customary  to  use  a  slide  provided  with  Zaj)pert's 
modified  ruling  (Fig.  73,  p.  211).  This  method  is  rather  more  accurate 
than  the  older  one  of  counting  the  leucocytes  in  a  separate  specimen 


BLOOD.  211 


of  blood.  Furthermore,  it  is  obviously  preferable  to  count  both  the 
erythrocytes  and  the  leucocytes  from  the  same  blood  sample.  To 
insure  accuracy  the  number  of  leucocytes  within  the  whole  ruled  region 


Tig.  73. — Zappert's  Modified  Ruling  of  Thoma-Zeiss  Counting  Chamber.     {Da 

Costa.) 

should  be  determined  in  duplicate  blood  samples.  This  includes 
the  examination  of  an  area  eighteen  times  as  great  as  the  old  style 
Thoma-Zeiss  central  ruling.  This  region  then  would  correspond 
to  3,600  of  the  small  squares  and,  if  duplicate  examinations  were  made, 
the  total  number  of  small  squares  examined  would  aggregate  7,200. 
The  calculation  would  be  as  follows: 

Number  of  leucocytes  in  7,200  .  .        .  .  Number  of  leucocytes  per  cubic 

•'  "         X  200X4,000-^7,200=  .„•      ,  ^ 

squares  '  '  millimeter. 

If  a  Zappert  slide  is  not  available,  a  good  plan  to  follow  is  to 
place  a  diaphragm  in  the  tube  of  the  ocular  of  the  microscope  consist- 
ing of  a  circle  of  black  cardboard  or  metaP  having  a  square  hole  in 
the  center  of  such  a  size  as  to  allow  of  the  examination  of  exactly 
100  squares  or  one-fourth  of  a  square  millimeter  at  one  time.  With 
this  arrangement  any  portion  of  the  specimen  may  be  examined  and 
counted  whether  within  or  without  the  ruled  area.  In  counting  by 
means  of  this  device  it  is,  of  course,  helpful  if  the  microscope  is  pro- 
vided with  a  mechanical  stage,  but  even  without  this  arrangement, 
if  the  observer  is  careful  to  see  that  the  leucocytes  at  the  extreme 
boundary  of  one  field  move  to  the  opposite  boundary  when  the  posi- 

'  Ehrlich's  mechanical  eye-piece  with  iris  diaphragm  is  also  very  satisfactor}^  for  this 
purpose. 


212  PHYSIOLOGICAL    CHEMISTRY. 

tion  of  the  slide  is  changed,  the  device  may  be  very  satisfactorily  em- 
ployed. The  leucocytes  should  be  counted  in  36  of  the  diaphragm- 
fields  in  duplicate  specimens  and  the  calculation  made  in  the  same  man- 
ner as  explained  above. 

If  the  leucocytes  are  counted  in  a  separate  specimen  of  blood 
ordinarily  the  diluting  fluid  is  0.3-0.5  per  cent  acetic  acid,  a  fluid  in 
which  the  leucocytes  alone  remain  visible.  Under  these  conditions 
the  dilution  is  customarily  made  in  the  pipette  having  11  as  the  final 
graduation.  The  capillary  portion  is  of  larger  caliber  and  so  rec[uires 
a  greater  amount  of  blood  to  fill  it  to  the  o.  5  or  i  mark  than  is  required 
in  the  use  of  the  other  form  of  pipette.  In  counting  the  leucocytes 
according  to  this  method  it  is  customary  to  draw  blood  into  the  pipette 
up  to  the  I  mark  and  immediately  fill  the  remaining  portion  of  the 
apparatus  to  the  11  graduation  with  the  0.3-0.5  per  cent  acetic  acid. 
It  then  remains  to  count  the  number  of  leucocytes  in  the  whole  central 
ruled  portion  of  400  squares.  This  should  be  done  in  duplicate 
samples  and  the  calculation  made  as  follows: 

Number   of    leucocytes   in   800  . .  ^       .  o  Number   of    leucocvtes   per   cubic 

X  4,000X10 -^000=  .,,•      .  •         ^ 

squares.  ^'  millimeter. 


CHAPTER  XIII. 

MILK.      . 

Milk  is  the  most  satisfactory  individual  food  material  elaborated 
by  nature.  It  contains  the  three  nutrients,  protein,  fat,  and  carbo- 
hydrate and  inorganic  salts  in  such  proportion  as  to  render  it  a  very 
acceptable  dietary  constituent.  It  is  a  specific  product  of  the  secre- 
tory activity  of  the  mammary  gland.  It  contains,  as  the  principal 
solids,  tri-olein,  tri-palmitin,  tri-stearin,  tri-hutyrin,  caseinogen,  lact- 
albiimin,  lac  to- globulin,  lactose,  and  calcium  phosphate.  It  also  contains 
at  least  traces  of  lecithin,  cholesterol,  urea,  creatine,  creatinine,  and  the 
tri-glycerides  of  caproic,  lauric,  and  myristic  acids.  Citric  acid  is  also 
said  to  be  present  in  milk  in  minute  quantity.  Fresh  milk  is  ampho- 
teric in  reaction  to  litmus,^  but  upon  standing  for  a  sufficiently  long 
time,  unsterilized,  it  becomes  acid  in  reaction,  due  to  the  production 
of  fermentation  lactic  acid, 

H     OH 

H-C-C-COOH, 

H     H 

from  the  lactose  contained  in  it.  This  is  brought  about  through 
bacterial  activity.  The  white  color  is  imparted  to  the  milk  partly 
through  the  fine  emulsion  of  the  fat  and  partly  through  the  medium 
of  the  caseinogen  in  solution.  The  specific  gravity  of  milk  varies 
somewhat,  the  average  being  about  i .  030.  Its  freezing-point  is 
about  — o.  56°  C. 

Fresh  milk  does  not  coagulate  on  being  boiled  but  a  film  con- 
sisting of  a  combination  of  caseinogen  forms  on  the  surface.  If 
the  film  be  removed,  thus  allowing  a  fresh  surface  to  come  in  contact 
with  the  air,  a  new  film  will  form  indefinitely  upon  the  application  of 
heat.  Surface  evaporation  and  the  presence  of  fat  facilitate  the  for- 
mation of  the  film,  but  are  not  essential  (Rettger).  As  Jamison  and 
Hertz  have  shown,  a  similar  film  will  form  on  heating  any  protein 

^  Human  milk  as  well  as  cow's  milk.     It  is,  however,  acid  to  phenolphthalein. 

21^ 


214  PHYSIOLOGICAL    CHEMISTRY. 

solution  containing  fat  or  paraffin.  If  the  milk  is  acid  in  reaction, 
through  the  inception  of  lactic  acid  fermentation,  or  from  any  other 
cause,  no  film  will  form  when  heat  is  applied,  but  instead  a  true  coagu- 
lation will  occur.  When  milk  is  boiled  certain  changes  occur  in  its 
odor  and  taste.  These  changes,  according  to  Rettger,  are  due  to  a 
partial  decomposition  of  the  milk  proteins  and  are  accompanied  by 
the  liberation  of  a  volatile  sulphide,  probably  hydrogen  sulphide. 

The  milk-curdling  enzymes  of  the  gastric  and  the  pancreatic 
juice  have  the  power  of  splitting  the  caseinogen  of  the  milk,  through 
a  process  of  hydrolysis,  into  soluble  casein  and  a  peptone-like  body. 


Fig.  74. — Normal  Milk  and  Colostrum. 
a,  Normal  milk;  h.  Colostrum. 

This  soluble  casein  then  forms  a  comljinalion  with  the  calcium  of  the 
milk  and  an  insoluble  curd  of  calcium  casein  or  casein  results.  The 
clear  fluid  surrounding  the  curd  is  known  as  whey. 

The  most  pronounced  difference  between  human  milk  and  cow's 
milk  is  in  the  protein  content,  ahhough  there  are  also  dififerences 
in  the  fats  and  likewise  striking  biological  differences  difficult  to 
define  chemically.  Jt  has  been  shown  that  the  caseinogen  of  human 
milk  differs  from  the  caseinogen  of  cow's  milk  in  being  more  difficult 
to  precipitate  by  acid  or  coagulate  by  gastric  rennin.  The  casein, 
curd  also  forms  in  a  much  loo.ser  and  more  flocculent  manner  than 
that  from  cow's  milk  and  is  for  this  reason  much  more  easily  digested 
than  the  latter.  Interesting  data  relative  to  the  comjxjsition  of  milk 
from  various  sources  may  be  gathered  from  the  following  table  which 
was  compiled  mainly  from  the  results  of  investigations  by  Jiunge  and 
by  Abderhalden.     It  will  bc  noted  that  the  composition  of  the  milk 


MILK. 


21! 


varies  directly  with  the  length  of  time  needed  for  the  young  of  the 
particular  species  to  double  in  weight. 


Species. 


Period  in  which 
Weight  of  the 
New-born  is 

Doubled  (Days). 


I  GO  Parts  of  Milk  Contain 


Proteins. 


Salts. 


Calcium. 


Phosphoric 
i      Acid. 


Man    . 
Horse 
Cow  . . 
Goat . . 
Sheep 
Pig  .  . . 
Cat    .. 
Dog 
Rabbit 


i8o 
60 

47 
22 

IS 
14 

9-5 

9 

6 


1.6 


.047 


2.0 

0.4 

0.124 

0.131 

3-5 

0.7 

0.160 

0.197 

3-7 

o.S 

0.197 

0.284 

4.9 

0.8 

0.245 

0.293 

5-2 

0.8 

0.249 

0.308 

7.0 

I.O 

7-4 

1-3 

0-455 

0.508 

10.4 

2-5 

0.891 

0.997 

Lactose,  the  principal  carbohydrate  constituent  of  milk,  is  an 
important  member  of  the  disaccharide  group.  It  occurs  only  in 
milk,  except  as  it  is  found  in  the  urine  of  women  during  pregnancy, 


Fig.  7; 


-L.-iCTOSE. 


during  the  nursing  period,  and  soon  after  weaning;  it  also  occurs 
in  the  urine  of  normal  persons  after  the  ingestion  of  a  very  large  amount 
of  lactose  in  the  food.  It  is  not  derived  directly  from  the  blood,  but  is 
a  specific  product  of  the  cellular  activity  of  the  mammary  gland.  It 
has  strong  reducing  power,  is  dextro-rotatory,  and  forms  an  osazone 
with  phenylhydrazine.  The  souring  of  milk  is  due  to  the  formation 
of  lactic  acid  from  lactose  through  the  agency  of  the  bacterium  lactis. 


2l6  PHYSIOLOGICAL    CHEMISTRY. 

Putrefactive  bacteria  in  the  alimentary  canal  may  bring  about  this 
same  reaction.  Lactose  is  not  fermentable  by  pure  yeast.  It  was 
recently  claimed  that  lactosbu  a  new  carbohydrate,  had  been  isolated 
from  milk. 

Caseinogen,  the  principal  protein  constituent  of  milk,  belongs  to 
the  group  of  phosphoproteins.  It  has  acidic  properties  and  com- 
bines with  bases  to  produce  salts.  It  is  not  coagulable  upon  boiling 
and  is  precipitated  from  its  neutral  solution  by  certain  metallic  salts 
as  well  as  upon  saturation  with  sodium  chloride  or  magnesium  sul- 
phate.    Its  acid  solution  is  precipitated  by  an  excess  of  mineral  acid. 

Lactalbumin  and  lacto-globulin,  the  protein  constituents  of  milk, 
next  in  importance  to  caseinogen,  closely  resemble  serum  albumin 
and  serum  globulin  in  their  general  properties.  According  to  Wrob- 
lewski,  a  protein  called  opalisin  is  also  present  in  milk. 

Colostrum  is  the  name  given  to  the  product  of  the  mammary 
gland  secreted  for  a  short  time  before  parturition  and  during  the 
early  period  of  lactation  (see  Fig.  74,  p.  214).  It  is  yellowish  in  color, 
contains  more  solid  matter  than  ordinary  milk,  and  has  a  higher 
specific  gravity  (i. 040-1. 080).  The  most  striking  diflerence  between 
colostrum  and  ordinary  milk  is  the  high  percentage  of  lactallnmiin 
and  lacto-globulin  in  the  former.  This  abnormality  in  the  protein 
content  is  responsible  for  the  coagulation  of  colostrum  upon  boiling. 

Such  enzymes  as  lipase,  amylase,  galactase,  catalase,  oxidases, 
peroxidases,  and  reductases  have  been  identified  in  milk,  but  not  all 
of  them  in  milk  of  the  same  species  of  animal. 

Among  the  principal  preservatives  used  in  connection  with  milk 
are  formaldehyde,  hydrogen  peroxide,  boric  acid,  borates,  salicylic 
acid,  and  salicylates. 

Experiments  on  Milk. 

1.  Reaction. — Test  the  reaction  of  fresh  cow's  milk  to  litmus. 

2.  Biuret  Test.  Make  the  biuret  test  according  to  directions 
given   on   page  90. 

3.  Microscopical  Examination.  Examine  fresh  whole  milk, 
skimmed  or  cenlrifiif^aled  milk,  and  colostrum  under  the  microscope. 
Compare  the  microscopical  appearance  with  Fig.  74,  page  214. 

4.  Specific  Gravity.  Determine  the  specific  gravity  of  Ijoth 
whole  and  skimmed  milk  (see  p.  254J.  Which  ))()ss(,'sses  the  higher 
specific  gravity?     Explain  why  this  is  so. 

;.  Film  Formation.     Place    10  c.c.   of   milk    in   a   small    beaker 


MILK.  217 

and  boil  a  few  minutes.  Note  the  formation  of  a  film.  Remove  the 
film  and  heat  again.  Does  the  film  now  form?  Of  what  substance 
is  this  film  composed?  The  biuret  test  was  positive,  why  do  we  not 
get  a  coagulation  here  when  we  heat  to  boiling? 

6.  Coagulation  Test. — Place  about  5  c.c.  of  milk  in  a  test-tube, 
acidify  slightly  with  dilute  acetic  acid  and  heat  to  boiling.  Do  you 
get  any  coagulation  ?    Why  ? 

7.  Action  of  Hot  Alkali. — To  a  little  milk  in  a  test-tube  add  a 
few  drops  of  potassium  hydroxide  and  heat.  A  yellow  color  develops 
and  gradually  deepens  into  a  brown.  To  what  is  the  formation  of 
this  color  due? 

8.  Test  for  Chlorides. — To  about  5  c.c.  of  milk  in  a  test-tube 
add  a  few  drops  of  very  dilute  nitric  acid  to  form  a  precipitate.  Filter 
off  this  precipitate  and  test  the  filtrate  for  chlorides.  Does  milk  contain 
any  chlorides  ? 

9.  Guaiac  Test. — To  about  5  c.c.  of  w^ater  in  a  test-tube  add  3 
drops  of  milk  and  enough  alcoholic  solution  of  guaiac  (strength 
about  1:60)^  to  cause  a  turbidity.  Thoroughly  mix  the  fluids  by 
shaking  and  observe  any  change  which  may  gradually  take  place 
in  the  color  of  the  mixture.  If  no  blue  color  appears  in  a  short  time, 
heat  the  tube  gently  below  60°  C.  and  observe  whether  the  color 
reaction  is  hastened.  In  case  a  blue  color  does  not  appear  in  the 
course  of  a  few  minutes,  add  hydrogen  peroxide  or  old  turpentine, 
drop  by  drop,  until  the  color  is  observed.  Fresh  milk  will  frequently 
give  this  blue  color  when  treated  with  an  alcoholic  solution  of  guaiac 
without  the  addition  of  hydrogen  peroxide  or  old  turpentine.  See 
discussion  on  page  188. 

10.  Tests  to  Differentiate  Between  Raw  Milk  and  Heated 
Milk. — [a)  Kastle's  Peroxidase  Reaction. — The  peroxidase  reaction  of 
milk  is  founded  upon  the  fact  that  small  amounts  of  raw  milk  will 
induce  the  oxidation  of  various  leuco  compounds  by  hydrogen  perox- 
ide. This  reaction  has  been  used  in  a  practical  way  as  the  most 
convenient  means  of  differentiating  between  raw  milk  and  heated 
milk.  Many  substances  have  been  employed  for  this  purpose,  e.  g., 
guaiac,  paraphenylenediamine,  ortol,  amidol,  etc.  Kastle  has  found 
that  a  dilute  solution  of  "trikresol"^  acts  as  a  sensitizing  agent  in  the 
peroxidase  reaction  and  offers  the  following  test  which  is  based  upon 
this  fact:  To  2-^  c.c.  of  raw  milk  in  a  test-tube  add  0.1-0.3  c.c.  of  M/io 

'  Buckmaster  advises  the  use  of  an  alcoholic  solution  of  guaiaconic  acid  instead  of  an 
alcoholic  solution  of  guaiac  resin.     Guaiaconic  acid  is  a  constituent  of  guaiac  resin. 

-  "Trikresol"  is  the  trade  name  of  an  antiseptic  which  contains  the  three  cresols  in 
approximately  equal  proportions. 


2l8  PHYSIOLOGICAL   CHEMISTRY. 

hydrogen  peroxide  and  i  c.c.  of  a  i  per  cent  solution  of  "trikresol." 
A  slight  though  unmistakable  yellow  color  will  be  observed  to  develop 
throughout  the  solution. 

Repeat  the  test  using  milk  which  has  been  boiled  or  heated  to 
80°  C.  for  10-20  minutes,  and  cooled,  and  note  that  no  yellow  color 
is  produced. 

The  color  reaction  in  the  case  of  the  raw  milk  probably  results 
from  the  oxidation  of  the  cresols  by  the  hydrogen  peroxide.  The 
first  product  of  this  oxidation^  then  oxidizes  the  leuco  compound, 
when  such  is  present,  and  causes  the  color  observed. 

{h)  Wilkinson  and  Peters'  Test.^ — To  10  c.c.  of  the  milk  to  be 
tested  add  2  c.c.  of  a  4  per  cent  alcoholic  solution  of  benzidine,  suffi- 
cient acetic  acid  to  coagulate  the  milk  (usually  2-3  drops)  and  finally 
2  c.c.  of  a  3  per  cent  solution  of  hydrogen  peroxide.  Raw  milk  yields 
an  immediate  blue  color.  In  adding  the  peroxide  it  is  best  to  permit 
it  to  flow  slowly  down  the  wall  of  the  vessel  containing  the  mixture 
instead  of  allowing  it  to  mix  with  the  milk.  Milk  which  has  been 
heated  to  78°  C.  or  above  remains  unchanged. 

11.  Saturation  with  Magnesium  Sulphate. — Place  about  5  c.c. 
of  milk  in  a  test-tube  and  saturate  with  solid  magnesium  sulphate. 
What  is  this  precipitate  ? 

12.  Influence  of  Gastric  Rennin  on  Milk. — Prepare  a  series  of 
five  tubes  as  follows: 

{a)  5  c.c.  of  fresh  milk +  0.2  per  cent  HCl  (add  drop  by  drop  until 
a  precipitate  forms). 

{h)   5  c.c.  of  fresh  milk +5  drops  of  rennin  solution. 

(c)    5  c.c.  of  fresh  milk-h  10  drops  of  0.5  per  cent  NajCOg. 

id)  5  c.c.  of  fresh  milk-|- 10  drops  of  ammonium  oxalate. 

{e)  5  c.c.  of  fresh  milk+5  drops  of  0.2  per  cent  HCI. 

Now  to  each  of  the  tubes  (c),  {d)  and  {e)  add  5  drops  of  rennin 
solution.  Place  the  whole  series  of  five  tubes  at  40°  C.  and  after  10-15 
minutes  note  what  is  occurring  in  the  different  tubes.  Give  a  reason 
for  each  particular  result. 

13.  Preparation  of  Caseinogen.  Fill  a  large  jjcaker  one-third 
full  of  skimmed  (or  centrifugated)  milk  and  dilute  it  with  an  equal 
volume  of  water.  Add  dilute  hydrochloric  acid  until  a  flocculent 
precipitate  forms.  Stir  after  each  acidification  and  do  not  add  an 
excess  of  the  acid  as  the  precipitate  would  dissolve.  Allow  the  precipi- 
tate to  settle,  decant  the  supernatant  fluid,  and  reserve  it  for  use  in 

'  Probably  some  organic  peroxide  or  quinoid  compound. 

*  Wilkinson  and  Peters:     Z.  Nahr-Genussm.,  XVI,  No.  3,  p.  172. 


MILK.  219 

later  (14-16)  experiments.  Filter  off  the  precipitate  of  caseinogen 
and  remove  the  excess  of  moisture  by  pressing  it  between  filter  papers. 
Transfer  the  caseinogen  to  a  small  beaker,  add  enough  95  per  cent 
alcohol  to  cover  it  and  stir  for  a  few  moments.  Filter,  and  press  the 
precipitate  between  filter  papers  to  remove  the  alcohol.  Transfer 
the  caseinogen  again  to  a  small  dry  beaker,  cover  the  precipitate  with 
ether  and  heat  on  a  water-bath  for  ten  minutes,  stirring  continuously. 
Filter  (reserve  the  filtrate),  and  press  the  precipitate  as  dry  as  possible 
between  filter  papers.  Open  the  papers  and  allow  the  ether  to  evapo- 
rate spontaneously.  Grind  the  precipitate  to  a  powder  in  a  mortar. 
Upon  the  caseinogen  prepared  in  this  way  make  the  following  tests: 

{a)  Solubility. — Try  the  solubility  in  the  ordinary  solvents. 

{h)  Milhn's  Reaction. ■^-M.okt  the  test  according  to  the  directions 
given  on  page  88. 

(c)  Biuret  Test. — Make  the  test  according  to  directions  given  on 
page  90. 

{d)  Hopkins-Cole  Reaction. — Make  the  test  according  to  the  direc- 
tions given  on  page  89. 

(e)  Loosely  Combined  Sidphur. — Test  for  loosely  combined  sulphur 
according  to  the  directions  given  on  page  100. 

(/)  Fusion  Test  for  Phosphorus. — Test  for  phosphorus  by  fusion 
according  to  directions  given  on  page  247. 

14.  Coagulable  Proteins  of  Milk. — Place  the  filtrate  from  the 
original  caseinogen  precipitate  in  a  casserole  and  heat,  on  a  wire 
gauze,  over  a  free  flame.  As  the  solution  concentrates,  a  coagulum 
consisting  of  lactalbumin  and  lactoglobuUn  will  form.  Continue  to 
concentrate  the  solution  until  the  volume  is  about  one-half  that  of 
the  original  solution.  Filter  off  the  coagulable  proteins  (reserve  the 
filtrate)  and  test  them  as  follows: 

{a)  Millmi's  Reaction. — Make  the  test  according  to  the  directions 
given  on  page  88. 

(b)  Biuret  Test. — Make  the  test  according  to  the  directions  given 
on  page  90. 

(c)  Hopkins-Cole  Reaction. — Make  the  test  according  to  the  direc- 
tions given  on  page  89. 

15.  Detection  of  Calcium  Phosphate. — Evaporate  the  fihrate 
from  the  coagulable  proteins,  on  a  water-bath,  until  crystals  begin 
to  form.  It  may  be  necessary  to  concentrate  to  15  c.c.  before  any 
crystallization  will  be  observed.  Cool  the  solution,  filter  off  the  crystals 
(reserve  the  filtrate),  and  test  them  as  follows: 


2  20  PHYSIOLOGICAL    CHEMISTRY. 

(a)  Microscopical  Examination. — Examine  the  crystals  and  com- 
pare them  with  those  in  Fig.  76. 

ib)  Dissolve  the  crystals  in  nitric  acid.  Test  part  of  the  acid 
solution    for    phosphates.      Render   the    remainder   of   the    solution 

slightly  alkaline  with  ammonia,  then  acidify 
with  acetic  acid  and  add  ammonium  oxa- 
late. Examine  the  crystals  under  the 
microscope  and  compare  them  with  those 
in  Fig.  99,  p.  340. 
f.^       ^C  ^^B  16.   Detection   of    Lactose. — Concen- 

trate   the   filtrate   from   the  calcium  phos- 
FiG.  76. — Calcium  Phosph.\te.       ,  .....  ,., 

phate  until  it  is  of  a  syrup-like  consistency. 

Allow  it  to  stand  over  night  and  observe  the  formation  of  crystals  of 
lactose.     ]Make  the  following  experiments. 

{a)  Microscopical  Examination. — Examine  the  crystals  and  com- 
pare them  with  those  in  Fig.  75,  page  215. 

{b)  Fehling^s  Test. — Try  Fehling's  test  upon  the  mother  liquor. 

(c)  Phenylhydrazine  Test. — Apply  the  phenylhydrazinc  test  to  some 
of  the  mother  liquor  according  to  the  directions  given  on  page  23. 

17.  Milk  Fat. — {a)  Evaporate  the  ether  filtrate  from  the  case- 
inogen  (Experiment  13)  and  observe  the  fatty  residue.  The  milk 
fat  was  carried  down  with  the  precipitate  of  caseinogen  and  was 
removed  when  the  latter  was  treated  with  ether.  If  centrifugated 
milk  was  used  in  the  preparation  of  the  caseinogen  the  amount  of  fat 
in  the  ether  filtrate  may  be  very  small.  To  secure  a  larger  yield  of 
fat  proceed  according  to  directions  given  under  (/>)  below. 

ib)  To  25  c.c.  of  whole  milk  in  an  evaporating  dish  add  a  little 
sand  or  filter  paper  and  evaporate  the  fluid  to  dryness  on  a  water- 
bath.  Grind  or  break  up  the  residue  after  cooling  and  extract  with 
ether  in  a  flask.  Filter  and  remove  the  ether  from  the  filtrate  by  evapo- 
ration.    How  can  you  identify  fats  in  the  ethereal  residue? 

18.  Saponification  of  Butter. — Dissolve  a  small  amount  of  butter 
in  alcohol  made  strongly  alkaline  with  potassium  hydroxide.  Place 
the  alcoholic-potash  solution  in  a  casserole,  add  about  100  c.c.  of  water 
and  boil  for  10-15  minutes  or  iinlil  Ihc  odor  of  alcohol  cannot  be 
detected.  Place  the  casserole  in  a  h(;od  and  neutralize  the  solution 
with  sulphuric  acid.  Note  the  odor  of  volatile  fatty  acids,  particularly 
butyric    acid. 

19.  Detection  of  Preservatives.-  (a)  Formaldehyde 

I.  (Jallii  A(  id  'lest.  .\(  idify  30  c.c.  of  milk  with  2  c.c.  of  normal 
>ulphuric  acid  anrl  distil.     Add  0.2-0.3  ^■^-  ^'>^  '^  saturated   alcoholic 


MILK.  221 

solution  of  gallic  acid  to  the  first  5  c.c.  of  the  distillate,  then  incline 
the  test-tube  and  slowly  introduce  3-5  c.c.  of  concentrated  sulphuric 
acid,  allowing  it  to  run  slowly  down  the  side  of  the  tube.  A  green 
ring,  which  finally  changes  to  blue,  is  formed  at  the  juncture  of  the 
fluids.  This  is  claimed,  by  Sherman,  to  be  twice  as  delicate  as  either 
the  sulphuric  acid  or  the  hydrochloric  acid  test  for  formaldehyde. 

II.  Leach'' s  Hydrochloric  Acid  Test. — Mix  10  c.c.  of  milk  and 
10  c.c.  of  concentrated  hydrochloric  acid  containing  about  0.002 
gram  of  ferric  chloride  in  a  small  porcelain  evaporating  dish  or  cas- 
serole and  gradually  raise  the  temperature  of  the  mixture,  on  a  water- 
bath,  nearly  to  the  boiling-point,  with  occasional  stirring.  If  formal- 
dehyde is  present  a  violet  color  is  produced,  while  a  brown  color 
develops  in  the  absence  of  formaldehyde.  In  case  of  doubt  the  mixture, 
after  having  been  heated  nearly  to  the  boiling-point  for  about  one 
minute,  should  be  diluted  with  50-75  c.c.  of  water,  and  the  color  of 
the  diluted  fluid  carefully  noted,  since  the  violet  color  if  present  will 
quickly  disappear.  Formaldehyde  may  be  detected  by  this  test  when 
present  in  the  proportion  i  :  250,000. 

{h)  Salicylic  and  Salicylates. — Remont's  Method.^  Acidify  20 
c.c.  of  milk  with  sulphuric  acid,  shake  well  to  break  up  the  curd,  add 
25  c.c.  of  ether,  mix  thoroughly,  and  allow  the  mixture  to  stand.  By 
means  of  a  pipette  remove  5  c.c.  of  the  ethereal  extract,  evaporate 
it  to  dryness,  boil  the  residue  with  10  c.c.  of  40  per  cent  alcohol,  and 
cool  the  alcoholic  solution.  Make  the  volume  10  c.c,  filter  through 
a  dry  paper  if  necessary  to  remove  fat,  and  to  5  c.c.  of  the  filtrate, 
which  represents  2  c.c.  of  milk,  add  2  c.c.  of  a  2  per  cent  solution  of 
ferric  chloride.  The  production  of  a  purple  or  violet  color  indicates 
the  presence  of  salicylic  acid. 

This  test  may  form  the  basis  of  a  quantitative  method  by  diluting 
the  final  solution  to  50  c.c.  and  comparing  this  with  standard  solutions 
of  salicylic  acid.  The  colorimetric  comparisons  may  be  made  in  a 
Duboscq   colorimeter. 

(c)  Hydrogen  Peroxide. — Add  2-3  drops  of  a  2  per  cent  aqueous 
solution  of  para-phenylenediamine  hydrochloride  to  10-15  c.c.  of 
milk.  If  hydrogen  peroxide  is  present  a  blue  color  will  be  produced 
immediately  upon  shaking  the  mixture  or  after  allowing  it  to  stand 
for  a  few  minutes.  It  is  claimed  that  hydrogen  peroxide  may  be 
detected  by  this  test  when  present  in  the  proportion  i  :  40,000. 

{d)  Boric  Acid  and  Borates. — To  the  ash,  obtained  according  to 
the   directions   given   in    Experiment   4,    page    407,  add    2    drops  of 

^  Sherman's  Organic  Analysis,  p.  232. 


222  PHYSIOLOGICAL    CHEMISTRY. 

dilute  hydrochloric  acid  and  i  c.c.  of  water.  Place  a  strip  of  tur- 
meric paper  in  the  dish  and  after  allowing  it  to  soak  for  about  one 
minute  remove  it  and  allow  it  to  dry  in  the  air.  The  presence  of  boric 
acid  is  indicated  by  the  production  of  a  deep  red  color  which  changes 
to  green  or  blue  upon  treatment  with  a  dilute  alkali.  This  test  is  sup- 
posed to  show  boric  acid  when  present  in  the  proportion  i  :  Sooo. 


CHAPTER  XIV. 

EPITHELIAL  AND  CONNECTIVE  TISSUES. 

EPITHELIAL  TISSUE  (KERATIN). 

The  albuminoid  keratin  constitutes  the  major  portion  of  hair,  horn, 
hoof,  feathers,  nails,  and  the  epidermal  layer  of  the  skin.  There  is  a 
group  of  keratins  the  members  of  which  possess  very  similar  properties. 
The  keratins  as  a  group  are  insoluble  in  the  usual  protein  solvents  and 
are  not  acted  upon  by  the  gastric  or  pancreatic  juices.  They  all  respond 
to  the  xanthoproteic  and  Millon  reactions  and  are  characterized  by 
containing  large  amounts  of  sulphur.  Keratin  from  any  of  its  sources 
may  be  prepared  in  a  pure  form  by  treatment,  in  sequence,  with  arti- 
ficial gastric  juice,  artificial  pancreatic  juice,  boiling  alcohol,  and  boiling 
ether,  from  twenty-four  to  forty-eight  hours  being  devoted  to  each 
process. 

Experiments  on  Epithelial  Tissue. 

Keratin. 

Horn  shavings  or  nail  parings  may  be  used  in  the  experiments 
which  follow: 

1.  Solubility.- — Test  the  solubility  of  keratin  in  the  ordinary  solvents 
(see  p.  22). 

2.  Millon'' s  Reaction. 

3.  Xanthoproteic  Reaction. 

4.  Adamkiewicz's  Reaction. 

5.  Hopkins-Cole  Reaction. 

6.  Test  for  Loosely  Combined  Snip] iiir. 

CONNECTIVE  TISSUE. 
I.  WHITE  FIBROUS  TISSUE. 

The  principal  solid  constituent  of  white  fibrous  connective  tissue 
is  the  albuminoid  collagen.  This  body  is  also  found  in  smaller  per- 
centage in  cartilage,  bone,  and  ligament,  but  the  collagen  from  the 
various  sources  is  not  identical  in  composition.     In  common  with  the 

223 


224  PHYSIOLOGICAL    CHEMISTRY. 

keratins,  collagen  is  insoluble  in  the  usual  protein  solvents.  It  differs 
from  keratin  in  containing  less  sulphur.  One  of  the  chief  characteris- 
tics of  collagen  is,  according  to  Hofmeister,  the  property  of  being  hydro- 
lyzed  by  boiling  acid  or  water  with  the  formation  of  gelatin.  Emmett 
and  Gies  claim  that  under  these  conditions  there  is  an  intramolecular 
rearrangement  of  collagen  and  the  resultant  gelatin  is  consequently 
not  the  product  of  hydrolysis.  The  liberation  of  ammonia  from  the 
collagen  during  the  process  apparently  confirms  this  view.  Collagen 
gives  Millon's  reaction  as  well  as  the  xanthoproteic  and  biuret  tests. 

The  form  of  white  fibrous  tissue  most  satisfactory  for  general  experi- 
ments is  the  tendo  AchiUis  of  the  ox.  According  to  Buerger  and  Gies 
the  fresh  tissue  has  the  following  composition: 

Water 62.87% 

Solids 37-13 

Inorganic  matter o .  47 

Organic  matter 36 .  66 

Fatty  substance  (ether-soluble) i  .04 

Coagulable  protein 0.22 

Mucoid 1 .  28 

Elastin ^  •  63 

Collagen 31 .59 

Extractives,  etc o. go 

The  mucoid  mentioned  above  is  called  tendomucoid  and  is  a  gly- 
coprotein. It  possesses  properties  similar  to  those  of  other  connective 
tissue  mucoids,  e.  g.,  osseomucoid  and  chondromucoid. 

Gelatin,  the  body  which  results  from  the  hydrolysis  of  collagen 
(see  statement  of  Emmett  and  Gies  above),  is  also  an  albuminoid.  It 
responds  to  nearly  all  the  protein  tests.  Jt  differs  from  the  keratins 
and  collagen  in  being  easily  digested  and  absorbed.  Gelatin  is  not  a 
satisfactory  substitute  for  the  protein  constituents  of  a  normal  diet, 
however,  since  a  certain  portion  of  its  nitrogen  is  not  available  for  the 
uses  of  the  organism.  Gelatin  from  cartilage  differs  from  the  gelatin 
from  other  sources  in  containing  a  lower  percentage  of  nitrogen.  Tyro- 
sine and  tryptoj)hane  are  not  numbered  among  the  decomposition 
jjroducts  of  gelatin,  hence  it  docs  not  res])ond  to  Millon's  reacti(jn  or 
the  lI(jpkins-Cole  reaction. 

Experiments  on  Whiti':  Imhrous  Ti.s.sue. 

'j'he  lendo  AchiUis  of  the  ox  may  be  taken  as  a  satisfactory  tyjjc  of 
the  while  fibrous  connective  tissue. 

I.  Preparation  of  Tendomucoid. — Dissect  away  the  fascia  from 
about  the  tendon  and  cut  the  clean  tendon  into  small  pieces.     Wash  the 


EPITHELIAL  AND    CONNECTIVE    TISSUES.  225 

pieces  in  water,  changing  the  wash  water  often  in  order  to  remove  as 
much  as  possible  of  the  soluble  protein  and  inorganic  salts.  Transfer 
the  washed  pieces  of  tendon  to  a  flask  and  add  300  c.c.  of  half-saturated 
lime  water. ^  Shake  the  flask  at  intervals  for  twenty-four  hours. 
Filtefr  off  the  pieces  of  tendon  and  precipitate  the  mucoid  with  dilute 
hydrochloric  acid.  Allow  the  mucoid  precipitate  to  settle,  decant  the 
supernatant  fluid  and  filter  the  remainder.  Test  the  mucoid  as 
follows : 

(a)  Solubility .^-Try  the  solubility  in  the  ordinary  solvents  (see 
page  22). 

{b)  Biuret  Test. — First  dissolve  the  mucoid  in  potassium  hydroxide 
solution  and  then  add  a  dilute  solution  of  cupric  sulphate. 

{c)   Test  for  Loosely  Combined  Sulphur. 

{d)  Hydrolysis  of  Tendomucoid. — Place  the  remainder  of  the  mucoid 
•in  a  small  beaker,  add  about  30  c.c.  of  water  and  2  c.c.  of  dilute  hydro- 
chloric acid  and  boil  until  the  solution  becomes  dark  brown.  Cool 
the  solution,  neutralize  it  with  solid  potassium  hydroxide,  and  test 
by  Fehling's  test.  With  a  reduction  of  Fehling's  solution  and  a 
positive  biuret  test  what  do  you  conclude  regarding  the  nature  of 
tendomucoid  ? 

2.  Collagen. — This  substance  is  present  in  the  tendon  to  the 
extent  of  about  32  per  cent.  Therefore  in  making  the  following  tests 
upon  the  pieces  of  tendon  from  which  the  mucoid,  soluble  protein,  and 
inorganic  salts  were  removed  in  the  last  experiment,  we  may  consider 
the  tests  as  being  made  upon  collagen. 

(a)  Solubility. — Cut  the  collagen  into  very  fine  pieces  and  try  its 
solubility  in  the  ordinary  solvents  (see  page  22). 

(b)  Milloji's  Reaction. 
(r)  Biuret  Test. 

(d)  Xanthoproteic  Reaction. 

(e)  Hopkins-Cole  Reaction. 

(/)  Test  for  Loosely  Combined  Sulphur. — Take  a  large  piece  of  col- 
lagen in  a  test-tube  and  add  about  5  c.c.  of  potassium  hydroxide  solu- 
tion. Heat  until  the  collagen  is  partly  decomposed,  then  add  1-2  drops 
of  plumbic  acetate  and  again  heat  to  boiling. 

(g)  Formation  of  Gelatin  from  Collagen. — Transfer  the  remainder 
of  the  pieces  of  collagen  to  a  casserole,  fill  the  vessel  about  two-thirds 
full  of  water  and  boil  for  several  hours,  adding  water  at  intervals  as 
needed.  By  this  means  the  collagen  is  transformed  and  a  body  known 
as  gelatin  is  produced  (see  p.  224). 

'  Made  b)^  mixing  equal  volumes  of  saturated  lime  water  and  water  from  the  faucet. 
15 


2  26  PHYSIOLOGICAL    CHEMISTRY. 

3.  Gelatin. — On  the  gelatin  formed  from  the  transformation  of 
collagen  in  the  above  experiment  {g),  or  on  gelatin  furnished  by  the 
instructor  make  the  following  tests: 

(a)  Soliibility. — Try  the  solubility  in  the  ordinary  solvents  (see 
page  22)  and  in  liot  water. 

(b)  Millon's  Reaction. 

(f)  Hopkins-Cole  Reaction. — Conduct  this  test  according  to  the 
modification  given  on  page  99. 

(d)   Test  for  Loosely  Combined  Sidphnr. 

Make  the  following  tests  upon  a  solution  of  gelatin  in  hot  water: 
(a)  Precipitation  by  Mineral  Acids. — Is  it  precipitated  by  strong 
mineral  acids  such  as  concentrated  hydrochloric  acid  ? 

{b)  Salting-out  Experiment.— SsituTate  a  little  of  the  solution  with 
solid  ammonium  sulphate.  Is  the  gelatin  precipitated?  Repeat  the 
experiment  with  sodium  chloride.     What  is  the  result  ? 

(c)  Precipitation  by  Metallic  Salts. — Is  it  precipitated  by  metallic 
salts  such  as  cupric  sulphate,  mercuric,  and  plumbic  acetate  ? 

(d)  Coagtdation  Test. — Does  it  coagulate  upon  boiling  ? 

(e)  Precipitation  by  Alkaloidal  Reagents. — Is  it  precipitated  by 
such  reagents  as  picric  acid,  tannic  acid,  and  trichloracetic  acid  ? 

(/)  Biuret  Test. — Does  it  respond  to  the  biuret  test  ? 

(g)  Bardach's  Reaction. — Does  it  yield  the  typical  crystals  of  this 
reaction?     (See  page  92.) 

(h)  Precipitation  by  Alcohol.— FiW  a  test-tube  one-half  full  of  95 
per  cent  alcohol  and  pour  in  a  small  amount  of  concentrated  gelatin 
solution.  Do  you  get  a  precipitate?  How  would  you  prepare  pure 
gelatin  from  the  tendo  Acliillis  of  the  ox? 

11.  YELLOW  ELASTIC  TISSUE  (ELASTIN). 

The  ligamentum  nucha;  of  the  ox  may  be  taken  as  a  satisfactory 
type  of  the  yellow  elastic  connective  tissue.  The  princij)al  solid  con- 
stituent of  this  tissue  is  elastin,  a  member  of  the  albuminoid  group. 
In  common  with  the  keratins  and  collagen,  elastin  is  an  insoluble  body 
and  gives  the  protein  color  reactions.  It  difTers  from  keratin  princi- 
pally in  the  fact  that  it  may  be  digested  by  enzymes  and  that  it  con- 
tains a  very  small  amount  of  sulphur. 

Yellow  elastic  tissue  also  contains  mucoid  and  collagen  but  these 
are  present  in  much  smaller  amount  than  in  white  fibrous  tissue,  as 
may  be  seen  from  the  following  percentage  composition  of  the  fresh 
ligamentum  nucha-  of  the  ox  as  determined  by  Vandegrift  and  Gies: 


EPITHELIAL  AND    CONNECTIVE    TISSUES.  22  7 

Water 57-57% 

Solids     42 .  43 

Inorganic  matter    o  •  47 

Organic  matter 41 .  96 

Fatty  substance  (ether-soluble)    .; 1.12 

Coagulable  protein 0.62 

Mucoid    0.53 

Elastin 3^- -^7 

Collagen    7  •  23 

Extractives,  etc 0.80 


Experiments  on  Elastin. 

1.  Preparation  of  Elastin  (Richards  and  Gies). — Cut  the  liga- 
ment into  fine  strips,  run  it  through  a  meat  chopper  and  wash  the  finely 
divided  material  in  cold,  running  water  for  24-48  hours.  Add  an 
excess  of  half -saturated  lime  water  (see  note  at  the  bottom  of  p.  225) 
and  allow  the  hashed  ligament  to  extract  for  48-72  hours.  Decant  the 
lime  water,  remove  all  traces  of  alkali  by  washing  in  water  and  then 
boil  in  water  with  repeated  renewals  until  only  traces  of  protein  material 
can  be  detected  in  the  wash  water.  Decant  the  fluid  and  boil  the  liga- 
ment in  ID  per  cent  acetic  acid  for  a  few  hours.  Treat  the  pieces  with 
5  per  cent  hydrochloric  acid  at  room  temperature  for  a  similar  period, 
extract  again  in  hot  acetic  acid  and  in  cold  hydrochloric  acid.  Wash 
out  traces  of  acid  by  means  of  water  and  then  thoroughly  dehydrolyze 
by  boiling  alcohol  and  boiling  ether  in  turn.  Dry  in  an  air-bath  and 
grind  to  a  powder  in  a  mortar. 

2.  Solubility. — Try  the  solubility  of  the  finely  divided  elastin, 
prepared  by  yourself  or  furnished  by  the  instructor,  in  the  ordinary 
solvents  (see  page  22).  How  does  its  solubility  compare  with  that  of 
collagen  ? 

3.  Millon's  Reaction. 

4.  Xanthoproteic  Reaction. 

5.  Biuret  Test. 

6.  Hopkins-Cole  Reaction. — Conduct  this  test  according  to  the 
modification  given  on  page  99. 

7.  Test  for  Loosely  Combined  Sulphur. 

III.  CARTILAGE. 

The  principal  solid  constituents  of  the  matrix  of  cartilaginous 
tissue  are  chondromucoid,  chondroitin-sulphuric  acid,  chondroalbiimoid 
and  collagen.     Chondromucoid  differs  from  the  mucoids  isolated  from 


228  PHYSIOLOGICAL    CHEMISTRY. 

Other  connective  tissues  in  the  large  amount  of  chondroitin-sulphuric 
acid  obtained  upon  decomposition.  Besides  being  an  important  con- 
stituent of  all  forms  of  cartilage,  chondroitin-sulphuric  acid  has  been 
found  in  bone,  ligament,  the  mucosa  of  the  pig's  stomach,  the  kidney 
of  the  ox,  the  inner  coats  of  large  arteries  and  in  human  urine.  It  may 
be  decomposed  through  the  action  of  acid  and  yields  a  nitrogenous 
body  known  as  chondroitin  and  later  this  body  yields  chondrosin. 
Chondrosin  is  also  a  nitrogenous  body  and  has  the  power  of  reducing 
Fehling's  solution  more  strongly  than  dextrose.  Sulphuric  acid  is  a 
bv-product  in  the  formation  of  chondroitin,  and  acetic  acid  is  a  by- 
product in  the  formation  of  chondrosin. 

Chondroalbumoid  is  similar  in  some  respects  to  elaslin  and  keratin. 
It  differs  from  keratin  in  being  soluble  in  gastric  juice  and  in  containing 
considerably  less  sulphur  than  any  member  of  the  keratin  group.  It 
gives  the  usual  protein  color  reactions. 

Experiments  on  C.-vrtilage. 

i.  Preparation  of  the  Cartilage.  Boil  the  trachea  of  an  ox  in 
water  until  the  cartilage  rings  may  be  completely  freed  from  the  sur- 
rounding tissue.  Use  the  cartilage  so  obtained  in  the  following 
experiments: 

2.  Solubility. — Cut  one  of  the  rings  into  \ery  small  pieces  and  try 
the  solubility  of  the  cartilage  in  the  ordinary  sohents  (see  page  22). 

3.  Millon's  Reaction. 

4.  Xanthoproteic  Reaction. 

5.  Hopkins-Cole  Reaction. — Conducl  this  test  according  to  the 
modification  gi\en  on  page  gc). 

6.  Test  for  Loosely  Combined  Sulphur. 

7.  Preparation  of  Cartilage  Gelatin.  Cut  the  remaining  carti- 
lage rings  into  small  pieces,  place  them  in  a  casserole  with  water  and 
boil  for  several  hours.  Filter  while  the  solution  is  still  hot.  Observe 
that  the  filtrate  soon  becomes  more  or  less  solid.  What  is  the  reason 
for  this?  Paring  a  ])orlion  of  ihc  material  into  solulion  l)\'  lical  and  try 
the  following  tests: 

(a)  Biuret  Test. 

(b)  Bardach's  Reaction. 

(c)  Test  for  Loosely  Combined  Sulphur. 

(d)  To  about  5  c.c.  of  the  solution  in  a  test-tube  add  a  few  drops  of 
barium  chloride.  Do  you  get  a  })reci})itatc,  and  if  so  to  what  is  the 
jjrec  ipilate  due  ? 


EPITHELIAL  AND    CONNECTIVE    TISSUES.  229 

(e)  To  about  5  c.c.  of  the  solution  in  a  test-tube  add  a  few  drops  of 
dilute  hydrochloric  acid  and  boil  for  a  few  moments.  Now  add  a  little 
barium  chloride  to  this  solution.  Is  the  precipitate  any  larger  than 
that  obtained  in  the  preceding  experiment  ?     Why  ? 

(/)  To  the  remainder  of  the  solution  add  a  little  dilute  hydrochloric 
acid  and  boil  for  a  few  moments.  Cool  the  solution,  neutralize  with 
solid  potassium  hydroxide,  and  try  Fehling's  test.     Explain  the  result. 

IV.   OSSEOUS  TISSUE. 

Bone  is  composed  of  about  equal  parts  of  organic  and  inorganic 
matter.  The  organic  portion,  called  ossein,  may  be  obtained  by 
removing  the  inorganic  salts  through  the  medium  of  dilute  acid. 
Ossein  is  practically  the  same  body  which  is  termed  collagen  in  the 
other  connective  tissues,  and  in  common  with  collagen  yields  gelatin 
upon  being  boiled  with  dilute  mineral  acid. 

In  common  with  the  other  connective  tissues  bone  contains  a 
mucoid  and  an  albumoid.  Because  of  their  origin  these  bodies  are 
called  osseomucoid  and  osseoalbumoid.  Osseomucoid,  when  boiled 
with  hydrochloric  acid,  yields  sulphuric  acid  and  a  substance  capable  of 
reducing  Fehling's  solution.  The  composition  of  osseomucoid  is  very 
similar  to  that  of  tendomucoid  and  chondromucoid  (see  page  104). 

Experiment  on  Osseous  Tissue. 

Analysis  of  Bone  Ash. — Take  i  gram  of  bone  ash  in  a  small 
beaker  and  add  a  little  dilute  nitric  acid.  What  does  the  effervescence 
indicate?  Stir  thoroughly  and  when  the  major  portion  of  the  ash  is 
dissolved  add  an  equal  volume  of  water  and  filter.  To  the  acid  filtrate 
add  ammonium  hydroxide  to  alkaline  reaction.  A  heavy  white  pre- 
cipitate of  phosphates  results.  (What  phosphates  are  precipitated 
here  by  the  ammonia?)  Filter  and  test  the  filtrate  for  chlorides,  sul- 
phates, phosphates,  and  calcium.  Add  dilute  acetic  acid  to  the 
precipitate  on  the  paper  and  test  this  filtrate  for  calcium  and  phos- 
phates. To  the  precipitate  remaining  undissolved  on  the  paper  add 
a  little  dilute  hydrochloric  acid  and  test  this  last  filtrate  for  phosphates 
and  iron. 

Reference  to  the  followins;  scheme  may  facilitate  the  analysis. 


230  PHYSIOLOGICAL    CHEMISTRY. 

BONE  ASH. 

I 

Add  dilute  nitric  acid,  stir  thoroughly  and  after  the  major  portion  of  the  ash  has  been 
brought  into  solution  add  a  little  distilled  water  and  filter. 


Residue  I. 


Filtrate  I. 


(discard)  Add  ammonium  hydroxide 

to  alkaline  reaction  and  filter. 


Residue  II. 

Treat  on  paper  with  acetic  acid. 


Residue  III. 

Treat  on  paper  with  hydro- 
chloric acid. 


Filtrate  IV. 
Test  for: 

1.  Iron. 

2.  Phosphates. 


Filtrate  III. 

Test  for: 

1.  Phosphates. 

2.  Calcium. 


Filtrate  II. 

Test  for: 

1.  Chlorides. 

2.  Sulphates. 

3.  Phosphates. 

4.  Calcium. 


V.  ADIPOSE  TISSUE. 
For  discussion  and  experiments  see  the  chapter  on  Fats,  page  128. 


CHAPTER  XV. 
MUSCULAR  TISSUE. 

The  muscular  tissues  are  divided  physiologically  into  the  vol- 
untary (striated)  and  the  involuntary  (non-striated).  In  the  chem- 
ical examination  of  muscular  tissue  the  voluntary  form  is  generally 
employed.  Muscle  contains  about  25  per  cent  of  solid  matter,  of 
which  about  four-fifths  is  protein  material  and  the  remaining  one-fifth 
extractives  and  inorganic  salts. 

The  proteins  are  the  most  important  of  the  constituents  of  mus- 
cular tissue.  In  the  living  muscle  we  find  two  proteins,  myosinogen 
and  para-myosinogen.  These  may  be  shown  to  be  present  in  muscle 
plasma  expressed  from  fresh  muscles.  In  common  with  the  plasma 
of  the  blood  this  muscle  plasma  has  the  power  of  coagulating,  and 
the  clot  formed  in  this  process  is  called  myosin.  In  the  onset  of  rigor 
mortis  we  have  an  indication  of  the  formation  of  this  myosin  clot 
within  the  body.  The  relation  between  the  proteins  of  living  and  dead 
muscle  is  represented  graphically  by  Halliburton  as  follows: 

Proteins  of  the  living  muscle. 


Para-myosinogen  (25%).  Myosinogen  (75%). 

Soluble  myosin. 


Myosin. 
(The  protein  of  the  muscle  clot.) 

Of  the  total  protein  content  of  living  muscle  about  75  per  cent 
is  made  up  by  the  myosinogen  and  the  remaining  25  per  cent  is  para- 
myosinogen. These  proteins  may  be  separated  by  subjecting  the 
muscle  plasma  to  fractional  coagulation  in  the  usual  way.  Under 
these  conditions  the  para-myosinogen  is  found  to  coagulate  at  47°  C. 
and  the  myosinogen  to  coagulate  at  56°  C.  It  is  also  claimed  by  some 
investigators  that  it  is  possible  to  separate  these  two  proteins  by  the 
fractional  ammonium  sulphate  method,  but  the  possibility  of  making 

2^1 


2^2  PHYSIOLOGICAL    CHEMISTRY. 

an  accurate  separation  by  this  method  is  somewhat  doubtful.  It  is 
well  established  that  para-myosinogen  is  a  globulin  since  it  responds 
to  certain  of  the  protein  precipitation  tests  and  is  insoluble  in  water. 
Myosinogen,  on  the  contrary,  is  not  a  typical  globulin  since  it  is  sol- 
uble in  water.  It  has  been  called  a  pseudo-globulin.  Myosin  possesses 
the  globulin  characteristics.  It  is  insoluble  in  water  but  soluble  in 
the  other  protein  solvents  and  is  precipitated  from  its  solution  upon 
saturation  with  sodium  chloride. 

X'cry  recently  Mellanby  has  reported  obser\'ations  which  he  claims 
indicate  that  there  is  only  one  protein  in  muscle  and  that  rigor  mortis 
is  due  to  the  coagulation  of  this  protein  under  the  combined  influences 
of  the  salt  present  in  the  muscle  and  the  lactic  acid  developed  upon 
the  death  of  the  muscle.  He  further  states  that  the  disappearance  of 
rigor  is  due  to  the  fact  that  the  lactic  acid  which  is  continually  formed 
brings  this  protein  into  solution. 

Under  the  name  exlraclives  we  class  a  numl^er  of  muscle  con- 
stituents which  occur  in  traces  in  the  tissue  and  may  be  extracted  by 
water,  alcohol,  or  ether.  There  are  two  classes  of  these  extractives, 
the  non-nitrogenous  extractives  and  the  nitrogenous  extractives.  Grouped 
under  the  non-nitrogenous  bodies  we  have  glycogen,  dextrin,  sugars, 
lactic  acid,  inosite,  CgHy(OH)g,  and  fat.  In  the  class  of  nitrogenous 
extractives  we  have  creatine,  creatinine,  xanthine,  hypoxanthine,  uric 
acid,  urea,  carnine,  guanine,  phosphocarnic  acid,  inosinic  acid,  carno- 
sine,  taurine,  carnitine,  novaine,  ignotine,  neosine,  oblitine,  carnomus- 
carine  and  methyl guanidine  (see  formulas  on  page  236).  Not  all  of 
these  extractives  are  present  in  the  muscles  of  all  species  of  animals. 
Other  extractives  besides  those  enumerated  above  have  been  de- 
scribed and  there  are  undoubtedly  still  others  whose  presence  remains 
undetermined.  A  detailed  consideration  would,  howe\'er,  be  un- 
profitable in  this  place. 

Glycogen  is  an  important  constituent  of  muscle.  The  content 
of  this  polysaccharide  in  muscle  varies  and  is  markedly  decreased 
by  intense  muscular  activity.  It  is  transformed  into  sugar  and  used 
as  fuel.  The  li\cr  is  the  organ  which  stores  the  reser\'e  suj)])ly  of 
glycogen  and  transforms  it  into  dextrose  which  is  passed  into  the 
blood  stream  and  so  carried  t(j  the  working  muscle  where  it  is  synthe- 
sized into  glycogen.  The  glycogen  thus  formed  is  then  changed  into 
dextrose  as  the  working  muscle  may  need  it. 

Glycogen  is  a  yjolysaccharide  and  has  the  same  percentage  com- 
position as  starch  and  dextrin.  It  resembles  starch  in  forming  an 
opalescent  sohiti<jn   and    resembles  dcxlrin    in   being  \'ery  sokible,   in 


MUSCULAR    TISSUE.  233 

giving  a  reddish  color  with  iodine  and  in  being  dextro-rotatory.  Gly- 
cogen may  be  prepared  from  muscle  by  extracting  with  boiling  water 
and  then  precipitating  the  glycogen  from  the  aciueous  solution  by  alco- 
hol: dilute  or  concentrated  potassium  hydroxide  may  also  be  used  to 
extract  the  glycogen.  Glycogen  may  be  prepared  in  the  form  of  a  white, 
tasteless,  amorphous  powder.  It  is  completely  precipitated  from  its 
solution  by  saturation  with  solid  ammonium  sulphate,  but  is  not  precipi- 
tated by  saturation  with  sodium  chloride.  It  may  also  be  precipitated 
by  alcohol,  tannic  acid,  or  ammoniacal  basic  lead  acetate.  It  has  the 
power  of  holding  cupric  hydroxide  in  solution  in  alkaline  fluids  but 
cannot  reduce  it.  It  may  be  hydrolyzed  with  the  formation  of 
dextrose  by  dilute  mineral  acids  and  is  readily  digested  by  amylolytic 
enzymes. 

Mendel  and  Leavenworth  have  recently  drawn  the  conclusion, 
from  the  examination  of  embryo  pigs,  that  embryonic  structures 
do  not  contain  exceptionally  large  amounts  of  glycogen.  The  dis- 
tribution of  the  glycogen  was  not  observed  to  differ  from  that  in  the 
adult  animal  except  that  the  liver  of  the  embryo  does  not  assume  its 
glycogen-storing  function  early.  They  further  draw  the  conclusion 
that  the  metabolic  transformations  of  glycogen  in  the  embryo  and  the 
adult  are  entirely  analogous. 

The  lactic  acid  occurring  in  the  muscular  tissue  of  vertebrates  is 
paralactic  or  sarcolactic  acid, 

H    OH 
H-C-C-COOH. 
H    H 

The  reaction  of  an  inactive  living  muscle  is  alkaline,  but  upon  the 
death  of  the  muscle,  or  after  the  continued  activity  of  a  living  muscle, 
the  reaction  becomes  acid,  due  to  the  formation  of  lactic  acid.  There 
is  a  difference  of  opinion  regarding  the  origin  of  this  lactic  acid.  Some 
investigators  claim  it  to  arise  from  the  carbohydrates  of  the  muscle, 
while  others  ascribe  to  it  a  protein  origin. 

Among  the  nitrogenous  extractives  of  muscle,  those  which  are 
of  the  most  interest  in  this  connection  are  creatine  and  the  purine  bases, 
xanthine  and  hypoxanthine.  Creatine  is  found  in  varying  amounts 
in  the  muscles  of  different  species,  the  muscles  of  birds  having  shown 
the  largest  amount.  It  has  also  been  found  in  the  blood,  the  brain, 
in  transudates  and  in  the  thyroid  gland.     Creatine  may  be  crystallized 


234 


PHYSIOLOGICAL    CHEMISTRY. 


and  forms  colorless  rhombic  prisms  (Fig.  77,  below)  which  are  soluble 
in  warm  water  and  practically  insoluble  in  alcohol  and  ether.  Upon 
boiling  a  solution  of  creatine  with  dilute  hydrochloric  acid  it  is  dehy- 
drolyzed  and  its  anhydride  creatinine  is  formed.  The  theory  that 
the  creatine  of  ingested  meat  is  transformed  into  creatinine  and  excreted 
in  the  urine  has  been  proven  untenable  through  the  recent  researches 
of  Folin,  Klercker,  and  Wolf  and  Shaffer.  It  is  now  known  that 
under  normal  conditions  the  ingestion  of  creatine  in  no  way  influences 


Fig. 


-Creatine. 


the  excretion  of  creatinine.  In  the  case  of  Eck  fistula  dogs,  however, 
London  and  Bolyarskii^  found  ingested  creatine  to  increase  the  out- 
put of  creatinine  in  the  urine.  This  finding  is  of  importance  as 
throwing  light  upon  the  role  of  the  liver  in  creatine  and  creatinine 
metabolism.  In  this  connection  it  is  important  to  note  that  there  is 
no  normal  excretion  of  endogenous  (see  p.  267)  creatine,  a  statement 
proven  by  the  fact  that  if  no  creatine  be  ingested  none  will  be  excreted. 
Folin^  has  shown  that  the  main  bulk  of  ingested  creatine  is  retained  in 
the  body,  unless  the  diet  contains  a  large  amount  of  protein  material. 
Under  certain  pathological  conditions  the  urine  may  contain  endog- 
enous creatine  which  is  probably  deri\ed  from  the  catabolism  of 
muscular  tissue,  as  Benedict,  Mellanby,  and  Shaffer  have  suggested. 
Besides  being  a  normal  constituent  of  muscle,  xanthine  has  been 
found  in  the  i;rain,  sjjleen,  j^ancreas,  thymus,  kidneys,  testicles,  liver, 

'  London  and  Bolyarskii:  Zeil.  phys.  chem.,  LXII,  p.  465,  1909. 
-  lolin      I  fammarsten  Festschrift,  p.  15. 


MUSCULAR   TISSUE. 


-OD 


and  in  the  urine.  It  may  be  obtained  in  crystalline  form  (Fig.  78, 
below),  but  ordinarily  it  is  amorphous.  Xanthine  is  easily  soluble  in 
alkalis,  less  soluble  in  water  and  dilute  acids,  and  entirely  insoluble  in 
alcohol  and  ether. 

Hypoxanthine  occurs  ordinarily  in  those  tissues  and  tiuids  which 
contain  xanthine.  It  has  been  found,  unaccompanied  by  xanthine, 
in  bone  marrow  and  in  milk.  Unlike  xanthine  it  may  be  easily  crys- 
tallized in  the  form  of  small,  colorless  needles.  It  is  readily  soluble 
in  alkalis,  acids,  and  boiling  water,  less  soluble  in  cold  water  and  prac- 
tically insoluble  in  alcohol  and  ether. 


Fig.  78. — XxsTHi^TE. 
After  the  drawings  of  Horbaczewski,  as  represented  in  Neubauer  and  \'ogeI.     {Ogden.) 

The  predominating  inorganic  salt  of  muscle  is  potassium  phos- 
phate. Besides  this  salt  we  have  present  chlorides  and  salts  of  sodium, 
calcium,  magnesium,  and  iron.     Sulphates  are  also  present  in  traces. 

Mendel  and  Saiki  have  recently  made  some  interesting  observa- 
tions upon  the  chemical  composition  of  non-striated  (involuntary) 
mammalian  muscle,  such  as  the  urinary  bladder  and  the  muscular 
coat  of  the  stomach  of  the  pig.  Hypoxanthine  was  found  to  be 
the  predominant  purine  base  present.  Creatine  and  paralactic  acid 
were  also  isolated.  These  investigators  were  unable  to  demonstrate, 
definitely,  the  presence  of  glycogen  in  the  non-striated  muscles  studied, 
but  state  that  "the  tissues  possess  the  property  of  transforming  glycogen 
in  the  characteristic  enzymatic  way."  The  most  important  part  of 
their  investigation  consists  in  a  rather  complete  analysis  of  the  inorganic 
constituents  of  these  muscles.  A  notable  difference  in  the  relative 
distribution  of  the  various  inorganic  constituents  was  observed,  a 
difference  which,  according  to  the  authors,  "can  be  accounted  for  in 


-^36 


PHYSIOLOGICAL    CHEMISTRY. 


part  only  by  an  admixture  of  lymph."  The  comparative  composition 
of  the  inorganic  portion  of  striated  and  non-striated  muscle  and 
of  blood  serum  for  comparison  is  shown  in  the  appended  table: 


K,0     Na,0 

Fe.Oj    CaO 

MgO 

CI       P.Os 

H,0 

Xon-striated  muscle  (Mendel 

and  Saiki)     

Skeletal  muscle  (Katz)    

Blood  serum  {.\bderhalden) .  . 

0.081 
0.306 

0.027 

0.328 
0.210 
0.425 

1 
o.oii    0.044 

0.008      O.OII 

0.012 

0.007 
0.047 

0.004 

0.I7I 
0.048 

0.363 

0.184 

0.487 

0.020; 

80.6 
72. q 

()i  .8 

Muscular  tissue  is  said  to  contain  a  reddish  pigment  called  myo- 
hcematin,  which  is  a  derivative  of  haemoglobin. 

The  so-called  "fatigue  substances"  of  muscle  are  carbon  diox- 
ide, paralactic  acid,  and  potassium  dihydrogen  phosphate. 

The  ordinary  commercial  "meat  extract"  is  composed  princi- 
pally of  the  water-soluble  constituents  of  muscle  and  contains  practi- 
cally nothing  of  nutritive  value.  The  protein  material  to  which  meat 
owes  its  value  as  an  article  of  diet  is  practically  all  removed  in  the 
preparation  of  the  extract. 

The  structural  formulas  of  the  nitrogenous  extractives  of  muscle 
are  as  follows: 


HN=C 


N.(CH3).CH,.COOH. 

Ckeati.ne,  CiHiiN.i02. 

M ethyl-gtianidine  acetic  acid. 


HN 


CO 


HN=C 


N.(CH).CH2 

Creatinine,  C4H7N.iO. 


Creatine  anhydride. 


NH2 

I 

C-0 

UREA.CON-.H, 


CH,.NH., 

I 
CH^.SO^OH 


Taurine.  CaHrNSOa. 

A  mino-cthyl-sulphonic  acid. 


o- 


(CH3)3.N 


\ 


-CO 


CH.,-CH  .  OH-CH, 

Carnitine,  C7H  if,N03. 
j^-trimcthyloxybutyrobetainc. 


MUSCULAR    TISSUE.  2^7 

Carnosine,  C„H,.N^O,.  > 

•'  9         14        4        3 

Neosine,  CgHTj^NOj.  . 

Novaine,  C7Hj7N02. 

Ignotine,  CyHj^N^Og. 

Phosphocarnic  acid,  CjoHj^NgO,  or  CjoH^jNgO.. 

Inosinic  add,  (HO)2.PO.O.CH2(CHOH)3.CH:(C5H3N,Oj.  . 

The  following  extractives  as  a  group  are  called  purine  bodies. 
Their  formulas,  together  with  that  of  purine  from  which  they  are 
derived  and  the  hypothetical  "purine  nucleus"  follow: 


N=CH 


^N-C^ 


HC     C-NH 


II        / 

N-C-N 

Purine,  CsHjNj. 

HN-CO 
HC     C-NH 


CH 


CH 


N-C-N 

Kypoxanthine,  C»H.iN40. 
6-oxypurine. 

HN-CO 


,C  'C^-N^ 


>C. 


3N-C,-N, 

Purine  Nucleus. 

HN-CO 
OC     C-NH 


CH 


HN-C-N 

Xanthine,  C5H4N4O2 
2— 6-dioxy  purine . 

N=C.NH. 


OC     C-NH 


>C0 


HN-C-NH 

Uric  Acid,  C5H4N-1O3. 
2—6— 8-trioxyptirine. 


HC     C-NH 


y 


CH 


N-C-N 

Adenine,  C.5H5N5 
b-aminopurine. 


HN-CO 


H.N.C     C-NH 


CH 


/ 

N-C-N 

Guanine,  ChHsN^O. 
2-amino—(>-oxy  purine . 

Experiments  on  Muscular  Tissue. 

I.  Experiments  on  "Living"  Muscle. 

I.  Preparation  of  Muscle  Plasma   (Halliburton).— Wash  out 
the  blood  vessels  of  a  freshly  killed  rabbit  with  0.9  per  cent  sodium 


23S  PHYSIOLOGICAL    CHEMISTRY. 

chloride.  This  can  best  be  done  by  opening  the  abdomen  and  in- 
serting a  cannula  into  the  aorta.  Xow  remo\e  the  skin  from  the 
lower  limbs,  cut  away  the  muscles  and  divide  them  into  very  small 
pieces  by  means  of  a  meat  chopper.  Transfer  the  pieces  of  muscle 
to  a  mortar  and  grind  them  with  clean  sand  and  a  little  5  per  cent 
magnesium  sulphate.  Filter  off  the  salted  muscle  plasma  and  make 
the  following  tests: 

{a)  Reaction. — Test  the  reaction  to  litmus.  What  is  the  reaction 
of  this  fresh  muscle  plasma  ? 

{b}  Fractional  Coagulation. — Place  a  little  muscle  plasma  in  a 
test-tube  and  arrange  the  apparatus  for  fractional  coagulation  as  ex- 
plained on  page  98.  Raise  the  temperature  very  carefully  from  30°  C. 
and  note  any  changes  which  may  occur  and  the  exact  temperature 
at  which  such  changes  take  place.  When  the  first  protein  (para- 
myosinogen) coagulates  filter  it  off  and  then  heat  the  clear  filtrate  as 
before,  being  careful  to  note  the  exact  temperature  at  which  the  next 
coagulation  (myosinogen)  occurs.  There  will  probably  be  a  pre- 
liminary opalescence  in  each  case  before  the  real  coagulation  occurs. 
Therefore  do  not  mistake  the  real  coagulation-point  and  filter  at  the 
wrong  time.  What  are  the  coagulation  temperatures  of  these  two 
proteins  ?     Which  protein  was  present  in  greater  amount  ? 

(c)  Formation  of  the  Myosin  Clot. — Dilute  a  portion  of  the  plasma 
with  3  or  4  times  its  volume  of  water  and  place  it  on  a  water-bath  or 
in  an  incubator  at  35°  C.  for  several  hours.  A  typical  myosin  clot  should 
form.  Note  the  muscle  serum  surrounding  the  clot.  Now  test  the 
reaction.  Has  the  reaction  changed,  and  if  so  to  what  is  the  change 
due?     Make  a  test  for  lactic  acid.     What  do  you  conclude? 

2.  Preparation  of  Muscle  Plasma  (v.  Furth). — Remove  the 
blood-frcc  muscles  of  a  rabbit  as  exj^laincd  above.  Finely  divide 
by  means  of  a  meat  chopper  and  grind  in  a  mortar  with  a  little 
clean  sand  and  some  0.9  per  cent  sodium  chloride.  Wrap  portions 
of  the  muscle  in  muslin  and  press  thoroughly  by  means  of  a  tincture 
press  or  lemon  squeezer.  Filter  and  make  the  tests  according  to  the 
flirections  gi\en  in  the  last  ex])crimcnt. 

3.  "Fuchsin-frog"  Experiment,  inject  a  saturated  aqueous 
solution  of  Fuchsin  "S"  into  the  lymi)h  sj)aces  of  a  frog  three  or 
four  times  daily  for  two  or  three  days,  in  this  way  thoroughly  satu- 
rating the  tissues  with  the  dye.  Pith  the  animal  (insert  a  heavy  wire 
or  blunt  needle  through  the  occipito  atlantoid  membrane),  remove 
the  skin  from  both  hind  legs  and  expose  the  sciatic  nerve  in  one  of 
them.     Insert  a  small  wire  hf)ok  through  the  jaws  of  the  frog  and 


MUSCULAR    TISSUE.  239 

suspend  the  animal  from  an  ordinary  clamp  or  iron  ring.  Pass  elec- 
trodes under  the  exposed  sciatic  nerve,  and  after  tying  the  other  leg 
to  prevent  any  muscular  movement,  stimulate  the  exposed  nerve  by 
means  of  make  and  break  shocks  from  an  induction  coil.  The 
stimulated  leg  responds  by  pronounced  muscular  contractions,  whereas 
the  tired  leg  remains  inactive.  Continue  the  stimulation  until  the 
muscles  are  fatigued.  The  muscular  activity  has  caused  the  pro- 
duction of  lactic  acid,  and  this  in  turn  has  reacted  with  the  injected 
fuchsin  to  cause  a  pink  or  red  color  to  develop.  The  muscles  of  the 
inactive  leg  still  remain  unchanged  in  color. 

The  normal  color  of  the  Fuchsin  "S"  when  injected  was  red, 
but  upon  being  absorbed  it  became  colorless  through  the  action  of 
the  alkalinity  of  the  blood.  Upon  stimulating  the  muscles,  how- 
ever, as  above  explained,  lactic  acid  was  formed  and  this  acid  reacted 
with  the  fuchsin  and  again  produced  the  original  color  of  the  dye. 

II.  Experiments  on  "Dead"  Muscle. 

I.  Preparation  of  Myosin. — Take  25  grams  of  finely  divided 
lean  beef  which  has  been  carefully  washed  to  remove  blood  and  lymph 
constituents  and  place  it  in  a  beaker  with  10  per  cent  sodium  chloride. 
Stir  occasionally  for  several  hours.  Strain  off  the  meat  pieces  by  means 
of  cheese  cloth,  filter  the  solution  and  saturate  it  with  sodium  chloride 
in  substance.  Filter  off  the  precipitate  of  myosin  and  make  the  tests 
as  given  below.  This  filtration  will  proceed  very  slowly.  Myosin 
collects  as  a  film  on  the  sides  of  the  filter  paper  and  may  be  removed 
and  tested  before  the  entire  volume  of  fluid  has  been  filtered.  If  this 
precipitate  remains  for  any  length  of  time  on  the  paper  in  contact 
with  the  air  it  will  become  transformed  into  the  protean  myosan.  Test 
the  myosin  precipitate  as  follows: 

{a)  Solubility. — Try  its  solubility  in  the  ordinary  solvents.  Is 
myosin  an  albumin  or  a  globulin  ? 

(b)  Xanthoproteic  Reaction. — See  page  89. 

(c)  Coagulation  Test. — Suspend  a  little  of  the  myosin  in  water 
in  a  test-tube  and  heat  to  boiling  for  a  few  moments.  Now  remove 
the  suspended  material  and  try  its  solubility  in  10  per  cent  sodium 
chloride.  What  property  does  this  experiment  show  myosin  to 
possess  ? 

Test  the  filtrate  from  the  original   myosin  precipitate  as  follows : 

(a)  Biuret  Test. — What  does  this  show? 

(b)  Place  a  little  of  the  solution  in  a  test-tube  and  heat  to  boiling. 


240  PHYSIOLOGICAL    CHEMISTRY. 

At  the  boiling-point  add  a  drop  of  dilute  acetic  acid  and  filter.  Test 
this  filtrate  for  proteose  with  picric  acid.  Is  any  proteose  present? 
Saturate  another  portion  of  the  filtrate  with  ammonium  sulphate 
and  test  for  peptone  in  the  usual  way  (see  page  112).  Do  you  find 
any  peptone?  From  your  experiments  on  "living"  and  "dead'' 
muscle  what  are  your  ideas  regarding  the  proteins  of  muscle? 

2.  Preparation  of  Glycogen. — Grind  a  few  scallops  in  a  mor- 
tar with  sand.  Transfer  to  an  evaporating  dish,  add  water,  and  boil 
for  20  minutes.  At  the  boiling-point  faintly  acidify  with  acetic  acid. 
Why  is  this  acid  added  ?  Filter,  and  divide  the  filtrate  into  two  parts. 
Xote  the  opalescence  of  the  solution.  Neutralize  or  make  faintly 
alkaline  one  portion  of  the  filtrate  and  test  it  as  follows: 

(a)  Iodine  Test. — To  5  c.c.  of  the  solution  in  a  test-tul)e  add  5-10 
drops  of  iodine  solution  and  2-;^  drops  of  10  per  cent  sodium  chloride. 
What  do  you  observe  ?  Is  this  similar  to  the  iodine  test  upon  any  other 
body  with  which  we  have  had  to  deal? 

(b)  Reduction  Test. — Does  the  solution  reduce  Fehling's  solution  ? 

(c)  Hydrolysis  of  Glycogen. — Add  10  drops  of  concentrated  hy- 
drochloric acid  to  10  c.c.  of  the  solution  and  boil  for  10  minutes.  Cool 
the  solution,  neutralize  with  solid  potassium  hydroxide  and  test  with 
Fehling's  solution.  Does  it  still  fail  to  reduce  Fehling's  solution?  If 
you  find  a  reduction  how  can  you  prove  the  identity  of  the  reducing 
substance  ? 

{d)  Influence  of  Saliva. — Place  5  c.c.  of  the  solution  in  a  test-tuljc, 
add  5  drops  of  saliva  and  place  on  the  water-l)ath  al  40^  C.  for  10 
minutes.     Does  this  now  reduce  Fehling's  solution? 

To  the  second  ]jarl  of  the  glyc(jgen  tiUrale  add  ,v  4  \()lumes  of 
95  per  cent  alcohol.  Allow  the  glycogen  ]jrecij)ilate  to  settle,  decant 
the  supernatant  fluid,  and  filter  the  remainder.  Heat  the  glycogen  on 
a  water-bath  to  remove  the  alcohol,  then  subject  it  to  the  following 
tests : 

(a)  Solubility. — Try  its  solubility  in  the  ordinary  soKents. 

(b)  Iodine  Test. — Place  a  small  amount  of  the  glycogen  in  a  de- 
pression of  a  test-tablet  and  add  2  3  (Iroj)s  of  dilute  iodine  solution 
and  a  trace  of  a  sodium  chloride  solution.  The  same  wine-red  color 
is  observed  as  in  the  iodine  tcsl  ujon  the  glycogen  sohilion. 

Separation  of  Extractives  from  Muscle. 

I.  Creatine,  ]>i.■^sol\•e  about  10  grams  of  a  (oninicrcial  extract 
of  meat  in   200  c.c.  of  warm   water.     Precii)ilate  the   inorganic  con- 


MUSCULAR    TISSUE. 


241 


stituents  by  neutral  lead  acetate,  being  careful  not  to  add  an  excess 
of  the  reagent.  Write  the  ec[uations  for  the  reactions  taking  place 
here.  Allow  the  precipitate  to  settle,  then  filter  and  remove  the 
excess  of  lead  in  the  warm  filtrate  by  hydrogen  sulphide.  Filter 
while  the  solution  is  yet  warm,  evaporate  the  clear  filtrate  to  a  syrup, 
and  allow  it  to  stand  at  least  48  hours  in  a  cool  place.  Crystals  of 
creatine  should  form  at  this  point.  Examine  under  the ,  microscope 
(Fig.  77,  page  234).  Treat  the  syrup  with  200  c.c.  of  88  per  cent 
alcohol,  stir  well  with  a  glass  rod  to  bring  all  soluble  material  into 
solution,  and  then  filter.  The  purine  bases  have  been  dissolved  and 
are  in  the  filtrate,  whereas  the  creatine  crystals  were  insoluble  in  the  88 
per  cent  alcohol  and  remain  on  the  filter  paper.  Wash  the  crystals  with 
88  per  cent  alcohol,  then  remove  them  and  bring  them  into  solution 
in  a  little  hot  water.     Decolorize  the  solution  by  animal  charcoal  and 


Fig.  79. — Hypoxanthine  Silver  Nitrate. 


concentrate  it  to  a  small  volume.  Allow  the  solution  to  cool  and  note 
the  separation  of  colorless  crystals  of  creatine.  Examine  these  crystals 
under  the  microscope  and  compare  them  with  those  reproduced  in 
Fig.  77'  page  234. 

2.  Hypoxanthine. — Evaporate  the  alcoholic  filtrate  from  the 
creatine  to  remove  the  alcohol.  Make  the  solution  ammoniacal  and 
add  ammoniacal  silver  nitrate  until  precipitation  ceases.  The  pre- 
cipitate consists  principally  of  hypoxanthine  silver  and  xanthine  silver. 
Collect  these  silver  salts  on  a  filter  paper  and  wash  them  with  water. 
Place  the  precipitate  and  paper  in  an  evaporating  dish  and  boil  for 
one  minute  with  nitric  acid  having  a  specific  gravity  of  i.i.  Filter 
16 


242 


PHYSIOLOGICAL    CHEMISTRY. 


while  hot  through  a  double  paper,  wash  with  the  same  strength  of 
nitric  acid  and  allow  the  solution  to  cool.  By  this  treatment  with 
nitric  acid  hypoxanthine  silver  nitrate  and  xanthine  silver  nitrate  have 
been  formed.  The  former  is  insoluble  in  the  cold  solution  and  sep- 
arates on  standing.  After  standing  several  hours  filter  off  the  hypox- 
anthine silver  nitrate  and  wash  with  water  until  the  wash-water  is  only 
slightly  acid  in  reaction.  Examine  the  crystals  of  hypoxanthine  silver 
nitrate  under  the  microscope  and  compare  them  with  those  in  Fig.  79, 
page  241.  Xow  wash  the  crystals  from  the  paper  into  a  beaker  with 
a  little  water  and  warm  the  liquid.  Remove  the  silver  by  hydrogen 
sulphide  and  filter.  By  this  means  hypoxanthine  nitrate  has  been 
formed  and  is  present  in  the  filtrate.  Concentrate  on  a  water-bath 
to  drive  off  hydrogen  sulphide  and  render  the  solution  slightly  alka- 
line with  ammonia.  Warm  for  a  time,  to  remove  the  free  ammonia, 
filter,  concentrate  the  filtrate  to  a  small  volume  and  allow  it  to  stand 
in  a  cool  place.  Hypoxanthine  should  crystallize  in  small  colorless 
needles.     Examine  the  crystals  under  the  microscope. 

3.  Xanthine. — To  the  filtrate  from  the  above  experiment  con- 
taining the  xanthine  silver  nitrate  add  ammonia  in  excess.  (The 
crystalline  form  of  xanthine  silver  nitrate  is  shown  in  Fig.  80,  below.) 


Fio.  80.  — Xanthin'k  .Sii.vkr  Nitratk. 


A  brownish-red  prccijjitale  of  xanthine  silver  forms.  Treat  this  sus- 
pended precipitate  with  hydrogen  sulphide  (do  not  use  an  excess  of 
hydrogen  sulphidej,  warm  the  mixture  for  a  few  moments  and  filter 
while  hot.  Concentrate  the  filtrate  to  a  small  vf)hime  and  put  away 
in  a  cool  place  for  crystallization  (Fig.  78,  p.  235J.  To  obtain  xan- 
thine in  crystalline  form  special  precautions  are  generally  ncces.sary. 


MUSCULAR    TISSUE.  243 

Evaporate  the  solution  to  dryness.  Make  the  following  tests  on  the 
crystals  or  residue: 

(a)  Xanthine  Test. — Place  about  one-half  of  the  crystalline  or 
amorphous  material  in  a  small  evaporating  dish,  add  a  few  drops  of 
concentrated  nitric  acid  and  evaporate  to  dryness  very  carefully  on  a 
water-bath.  The  yellow  residue  upon  moistening  with  caustic  potash 
becomes  red  in  color  and  upon  further  heating  assumes  a  purplish-red 
hue.  Now  add  a  few  drops  of  water  and  warm.  In  this  way  a  yellow 
solution  results  which  yields  a  red  residue  upon  evaporation.  How 
does  this  differ  from  the  Murexide  test  upon  uric  acid  ? 

(h)  WeideVs  Reaction. — By  gently  heating  bring  the  remainder 
of  the  xanthine  crystals  or  residue  into  solution  in  bromine-water. 
Evaporate  the  solution  to  dryness  on  a  water-bath.  Remove  the  stop- 
per from  an  ammonia  bottle  and  by  blowing  across  the  mouth  of  the 
bottle  direct  the  fumes  of  ammonia  so  that  they  come  in  contact  with 
the  dry  residue.  Under  these  conditions  the  presence  of  xanthine  is 
shown  by  the  residue  assuming  a  red  color.  A  som.ewhat  brighter  color 
may  be  obtained  by  using  a  trace  of  nitric  acid  with  the  bromine-water. 
By  the  use  of  this  modification,  however,  we  may  get  a  positive  reaction 
with  bodies  other  than  xanthine. 

Hurthle's  Experiment. 

Tease  a  very  small  piece  of  frog's  muscle  on  a  microscopical  slide. 
Expose  the  slide  to  ammonia  vapor  for  a  few^  moments,  then  adjust  a 
coverglass,  and  examine  the  muscle  fibers  under  the  microscope. 
Xote  the  large  number  of  crystals  of  ammonium  magnesium  phosphate, 

NH,-0 

\ 
Mg-0-P  =  0 

\/ 
O 

distributed  everyvrhere  throughout  the  muscle  fiber,  thus  demon- 
strating the  abundance  of  phosphates  and  magnesium  in  the  muscle 
(Fig.  96,  page    296.) 


CH.^PTER  XVI. 
NERVOUS   TISSUE. 

In  common  with  the  other  solid  tissues  of  the  body,  nervous  tissue 
contains  a  large  amount  of  water.  The  percentage  of  water  present 
depends  upon  the  particular  form  of  nervous  tissue  but  in  all  forms 
it  is  invariably  greater  in  the  gray  matter  than  in  the  white.  Embry- 
onic nervous  tissues  also  contain  a  larger  percentage  of  water  than 
the  tissues  of  adult  life.  The  gray  matter  of  the  brain  of  the  foetus, 
for  instance,  contains  about  92  per  cent  of  water,  whereas  the  gray 
matter  of  the  brain  of  the  adult  contains  but  83-84  per  cent  of  the 
fluid. 

Among  the  solid  constituents  of  nervous  tissue  are  proteins,  cho- 
lesterol, cerebrin,  lecithin,  kephalin,  protagon  {?),  paranucleoprotagon, 
niiclein,  neurokeratin,  collagen,  extractives,  and  inorganic  salts.  The 
proteins  are  present  in  the  greatest  amount  and  comprise  about  50 
per  cent  of  the  total  solids.  Three  distinct  proteins,  two  globulins, 
and  a  nucleoprotein,  have  been  isolated  from  nervous  tissue.  The 
globulins  coagulate  at  47°  C.  and  70-75°  C,  respecti\ely.  while  the 
nucleoprotein  coagulates  at  56-60°  C.  This  nucleoprotein  contains 
about  0.5  per  cent  of  phosphorus  (Halliburton,  Levene).  Nervous 
tissue  is  composed  of  a  relativ'ely  large  quantity  of  a  variety  of  com- 
pounds which  collectively  may  be  grouped  under  the  term  ''lipoid" — 
substances  resembling  the  fats  in  some  of  their  physical  properties 
and  reactions  but  distinct  in  their  composition.  We  will  class  cerebrin, 
cholesterol,  and  the  ph(js]jhorized  fats  as  "lipoids." 

The  groujj  of  phosphorized  fats  are  very  important  constituents  of 
nervous  tissue.  The  best  known  members  of  this  grou])  are  lecithin, 
protagon  {?)  and  kephalin.  Lecithin  occurs  in  larger  amount  than 
the  other  mem})ers  of  the  group,  has  been  more  thoroughly  studied 
than  the  others  and  is  apparently  of  greater  importance.  \'\)ov\  rierom 
[xjsition  lecithin  yields /rt//y  acid,  glycero- phosphor  i(  (i(/d,un(\  t  hoi  inc. 
Each  lecithin  molecule  contains  two  f;itt\'  a(  id  radicals  which  may  be 
those  of  the  same  or  different  fatty  acids.  Thus  we  ha\e  different 
lecithins  dejjcnding  ujjon  the  ])articular  fatly  acid  radicals  which  are 
present  in  the  molecule.  The  formula  of  a  typical  lc(  illiin  would  be 
the  following: 

244 


NERVOUS    TISSUE.  245 

CH,0-C,,H3,C0 
CHO  -C,,Il,,CO 

CH^O-PO-O-CH, 

\ 

(CH3)3^N 

/ 
OH  HO 

This  lecithin  would  be  called  distearyl-lecithin  or  choline-distearyl- 
glycero-phosphoric  acid.  Upon  decomposition  the  molecule  splits 
according  to  the  following  reaction: 

C,,H,,NPO,  +  3H,0  =  2  (C,,H3,0,)  +  C3H,PO,+  C.H^.NO,. 

Lecithin.  Stearic  acid.        Glycero-phosphoric        Choline. 

acid. 

The  lecithins  are  not  confined  to  the  nervous  tissues  but  are  found  in 
nearly  all  animal  and  vegetable  tissues.  Lecithin  is  a  primary  con- 
stituent of  the  cell.  It  is  soluble  in  chloroform,  ether,  alcohol,  benzene, 
and  carbon  disulphide.  The  chloroform  or  alcohol-ether  solution 
may  be  precipitated  by  acetone.  Lecithin  may  be  caused  to  crystal- 
lize in  the  form  of  small  plates  by  cooling  the  alcoholic  solution  to  a 
low  temperature.  It  has  the  powxr  of  combining  with  acids  and  bases, 
and  the  hydrochloric  acid  combination  has  the  power  of  forming  a 
double  salt  with  platinic  chloride. 

Choline,  as  was  indicated  above,  is  one  of  the  decomposition 
products  of  lecithin.  It  is  trimethyl-oxyethyl-ammoniiim  liydroxide  and 
has  the  following  formula: 

CH,.CH40H) 

N-(CH3), 
OH 


\ 

\ 


Recent  researches  have  shown  that  great  importance  is  to  be  attached 
to  the  detection  of  choline  in  the  ccrebro-spinal  fluid  and  the  blood  in 
certain  cases  of  degenerative  disease  of  the  nervous  system.  In  this 
connection  tests  for  choline  (see  p.  248)  are  of  interest  and  value. 

Protagon,  another  nitrogenous  phosphorized  substance  is  a  body 
over  which  there  has  been  much  discussion.  Upon  decomposition  it 
is  said  by  some  investigators  to  yield  cerebrin  and  the  decomposition 


246  PHYSIOLOGICAL    CHEMISTRY. 

products  of  lecithin.  It  has  recently  been  shown  by  Posner  and  Gies 
as  well  as  by  Rosenheim  and  Tebb  that  protagon  is  a  mixture  and  has 
no  existence  as  a  chemical  individual. 

Kephalin  is  the  third  member  of  the  group  of  phosphorized  fats. 
It  is  precipitated  from  its  acetone-ether  extract  by  alcohol.  It  contains 
about  4  per  cent  of  phosphorus  and  has  been  given  the  formula  C^jH^g- 
NPOj3.     Kephalin  may  be  a  stage  in  lecithin  metabolism. 

Cerebrin,  a  substance  containing  nitrogen  but  no  phosphorus,  is  an 
important  constituent  of  the  white  matter  of  nervous  tissue.  It  has 
also  been  found  in  the  spleen,  pus,  and  in  egg  yolk.  It  may  be  ex- 
tracted from  the  tissue  by  boiling  alcohol  and  is  insoluble  in  cold 
alcohol,  cold  and  hot  ether,  and  in  water  and  dilute  alkalis.  Cerebrin 
is  a  mixture  containing  phrenosin  (pseudo-cerebrin  or  cerebron),  a 
body  yielding  the  carbohydrate  galactose  on  decomposition. 

Cholesterol,  one  of  the  primary  cell  constituents,  is  present  in 
fairlv  large  amount  in  nervous  tissue.  It  is  a  mon-atomic  alcohol 
with  the  formula  C^^U^.OH.  It  was  formerly  called  a  "non-saponi- 
fiable  fat"  but  since  it  is  not  changed  in  any  way  by  boiling  alkalis  il 
is  not  a  fat.  It  is  soluble  in  ether,  chloroform,  benzene,  and  hot  alcohol. 
It  crystallizes  in  the  form  of  thin,  colorless^  transparent  plates  (Fig. 
42,  p.  155).  Cholesterol  occurs  abundantly  in  one  form  of  biliary 
calculus.  It  has  also  been  found  in  feces,  wool  fat,  egg  yolk,  and  milk, 
frequently  in  the  form  of  its  esters  of  higher  fatty  acids. 

Paranucleoprotagon  is  a  phosphorized  substance  originally  isolated 
from  brain  tissue  by  Ulpiani  and  Lelli  and  recently  reinvestigated  by 
Steel  and  Gies.    It  is  said  to  possess  lecithoprotein  characteristics. 

Nervous  tissue  yields  about  i  per  cent  of  ash  which  is  made  up  in 
great  part  oi  alkaline  phosphates  and  chlorides. 

.  Experiments  on  thk  Lipoids  or  Nkrvous  Tissue.^ 

I.  Preparation  of  Lecithin. — Treat  the  macerated  brain  of  a 
sheep  with  ether  and  allow  it  to  stand  in  the  cold  for  48-72  hours. 
The  cold  ether  will  extract  lecithin  and  cholesterol.  Filler  and  add 
acetone  to  the  filtrate  to  precipitate  the  lecithin.  Filler  off  the  lecithin 
and  test  it  as  follows: 

'  Preparation  of  So-callcfl  Protagon.— Macerate  the  brain  of  a  siieej),  treat  with  85 
per  cent  air  oho!  and  warm  on  a  water-bath  at  45°  C.  for  two  hours.  Filter  hot  into  a 
Ijottie  or  strong  flask  anri  cool  to  0°  C'.  for  one-lialf  hour  l)y  means  of  a  freezing  mixture. 
Hy  this  procefiure  both  protagon  and  cholesterol  are  causeil  to  precipitate.  Filter  the  cold 
volution  rapidly  and  treat  the  precij»itate  on  the  pjijjer  with  ice  cold  ether  to  dissolve  out 
the  cholesterol.  The  protagon  may  now  be  redissolved  in  warm  85  per  cent,  alcohol  from 
uhicli  soliitiot)  il  will  ])r((ipil.ilc  u[)ori  (ooling. 


NERVOUS    TISSUE.  247 

(a)  Microscopical  Examination. — Suspend  a  small  portion  in  a 
drop  of  water  on  a  slide  and  examine  under  the  microscope. 

(b)  Osmic  Acid  Test. — Treat  a  small  portion  with  osmic  acid. 
What   happens  ? 

(c)  Acrolein  Test. — Make  the  acrolein  test  according  to  directions 
on  page  132. 

{d)  ^^ Fusion"'  Test  for  Phosphorus. — Place  some  of  the  lecithin 
prepared  above  in  a  small  porcelain  crucible,  add  a  suitable  amount 
of  a  fusion  mixture  composed  of  potassium  hydroxide  and  potassium 
nitrate  (5:1)  and  heat  carefully  until  the  resulting  mixture  is  colorless. 
Cool,  dissolve  the  mass  in  a  little  warm  w^ater,  acidify  with  nitric 
acid,  heat  to  boiling,  and  add  a  few  cubic  centimeters  of  molybdic 
solution.  In  the  presence  of  phosphorus  a  yellow  precipitate  forms. 
What  is  it? 

2.  Preparation  of  Cholesterol. — Place  a  small  amount  of 
macerated  brain  tissue  under  ether  and  stir  occasionally  for  one  hour. 
Filter,  evaporate  the  filtrate  to  dryness  on  a  water-bath,  and  test  the 
cholesterol  according  to  directions  given  below.  (If  it  is  desired,  the 
ether  extract  from  the  so-called  protagon,  or  the  ether-acetone  filtrate 
from  the  lecithin  may  be  used  for  the  isolation  of  cholesterol.  In  these 
cases  it  is  simply  necessary  to  evaporate  the  solution  to  dryness  on  a 
water-bath.)  Upon  the  cholesterol  prepared  by  either  of  the  above 
methods  make  the  following  tests: 

{a)  Microscopical  Examination. — Examine  the  crystals  under  the 
microscope  and  compare  them  with  those  in  Fig.  42,  page  155. 

(b)  Iodine-sulphuric  Acid  Test. — Place  a  few  crystals  of  cho- 
lesterol in  one  of  the  depressions  of  a  test-tablet  and  treat  with  a  drop 
of  concentrated  sulphuric  acid  and  a  drop  of  a  very  dilute  solution  of 
iodine.  A  play  of  colors,  consisting  of  violet,  blue,  green,  and  red, 
results. 

(c)  The  Liebermann-Biir chard  Test. — Dissolve  a  few  crystals  of 
cholesterol  in  2  c.c.  of  chloroform  in  a  dry  test-tube.  Now  add  10 
drops  of  acetic  anhydride  and  1-3  drops  of  concentrated  sulphuric 
acid.  The  solution  becomes  red,  then  blue,  and  finally  bluish-green 
in  color. 

{d)  Salkowski^s  Test. — Dissolve  a  few  crystals  of  cholesterol  in  a 
little  chloroform  and  add  an  equal  volume  of  concentrated  sulphuric 
acid.  A  play  of  colors  from  bluish-red  to  cherry-red  and  purple  is 
noted  in  the  chloroform,  while  the  acid  assumes  a  marked  green 
fluorescence. 

(e)  Schiffs  Reaction. — To   a   little   cholesterol   in   an   evaporating 


248  PHYSIOLOGICAL    CHEMISTRY. 

dish  add  a  few  drops  of  SchifT's  reagent.^  E\-aporate  to  dryness  over 
a  low  flame  and  obser\-e  the  reddish-violet  residue  which  changes  to  a 
bluish- violet. 

( /)  Phosphorus. — Test  for  phosphorus  according  to  directions 
given  on  page  247.    Is  phosphorus  present? 

3.  Preparation  of  Cerebrin. — Treat  the  macerated  brain  tissue, 
in  a  flask,  with  95  per  cent  alcohol  and  boil  on  a  water-bath  for  one- 
half  hour,  keeping  the  volume  constant  by  adding  fresh  alcohol  as 
needed.  Filter  the  solution  hot  and  stand  the  cloudy  filtrate  away  for 
twenty-four  hours.  (If  the  filtrate  is  not  cloudy  concentrate  it  upon 
the  water-bath  until  it  is  so.)  Filter  off  the  cerebrin  and  test  it  as 
follows : 

(a)  Microscopical  Examination. — Suspend  a  small  portion  in  a 
drop  of  water  on  a  slide  and  examine  under  the  microscope. 

(b)  Solubility. — Try  the  solubility  of  cerebrin  in  the  usual  solvents 
and  in  hot  and  cold  alcohol  and  hot  and  cold  ether. 

(c)  Phosphorus. — Test  for  phosphorus  according  to  directions  on 
page  247.    How  does  the  result  compare  with  that  on  lecithin? 

(d)  Place  a  little  cerebrin  on  platinum  foil  and  warm.  Note  the 
odor. 

(e)  Hydrolysis  of  Cerebrin. — Place  the  remaining  cerebrin  in  a 
small  evaporating  dish,  add  equal  volumes  of  water  and  dilute  hydro- 
chloric acid,  and  boil  for  one  hour.  Cool,  neutralize  with  solid  potas- 
sium hydroxide,  filter,  and  test  with  Fehling's  solution.  Is  there  any 
reduction,  and  if  so  how  do  you  explain  it? 

4.  Tests  for  Choline. — (a)  Rosenheim's  Periodide  Test. — Pre- 
pare an  alcoholic  extract  of  the  Huid  under  examination,  and  after 
evaporation,  apply  Rosenheim's  iodo-potassium  iodide  solution^  to  a 
little  of  the  residue.  In  a  short  time  dark  brown  plates  and  prisms  of 
choline  periodide  begin  to  form  and  may  be  detected  by  means  of  the 
microscope.  Occasionally  they  are  large  enough  to  Ijc  \isiblc  to  the 
naked  eye.  They  somewhat  resemble  crystals  of  hiemin  (see  p.  i(;4). 
If  the  slide  be  jjcrmilted  to  stand,  thus  allowing  the  fluid  to  evaporate, 
the  crystals  will  disa};pear  and  Iea\c  brown  oily  drops.  They  will 
reajjjjear,  however,  upon  the  addition  of  fresh  iodine  solution,  v. 
Stanek  claims  that  this  choline  (onipound  has  the  formula 
C,H,,NOI.I,. 

ib)  Rosenheim's   Bismuth    Pest. — Kxlracl    llic    llnid    uucUt    exam- 

'  Schiff's  rcdjjcnl  consists  of  a  niixliiix-  of  three  volumes  of  (  (jik  cnl  latnl  siil|)liiii  ii 
acifl  anrl  one  vfdumc  of  jo  |jcr  cent  ferric   ( liloride. 

^  Preparefl  by  flissolving  2  f(ranis  of  iodine  and  6  f^ranis  of  |)ol;issiiiiii  iudidc  in  100  <  .1  . 
of  Uater. 


NERVOUS    TISSUE.  249 

ination  with  absolute  alcohol,  evaporate,  and  re-extract  the  residue. 
Repeat  the  extraction  several  times.  Dissolve  the  final  residue  in 
2-3  c.c.  of  water  and  add  a  drop  of  Kraut's  reagent.^  Choline  is 
indicated  by  the  appearance  of  a  bright  brick-red  precipitate. 

^  Dissolve  272  grams  of  potassium  iodide  in  water  and  add  80  grams  of  bismuth  sub- 
nitrate  dissolved  in  200  grams  of  nitric  acid  (sp.  gr.  i;i8).  Permit  the  potassium  nitrate 
to  crystallize  out,  then  filter  it  off  and  make  the  filtrate  up  to  i  liter  with  water. 


CHAPTER  XVII. 

URINE:  GENERAL    CHARACTERISTICS    OF    NORMAL    AND 
PATHOLOGICAL   URINE. 

Volume. — The  volume  of  urine  excreted  by  normal  indi\"iduals 
during  any  definite  period  fluctuates  within  very  wide  limits.  The 
average  output  for  twenty-four  hours  is  placed  by  German  writers 
between  1500  and  2000  c.c.  This  value  is  not  strictly  appHcable  to  con- 
ditions in  Amicrica,  however,  since  it  has  been  found  that  the  a^•erage 
normal  excretion  of  the  aduU  male  American  falls  within  the  lower 
values  of  1000-1200  c.c.  The  volume-excretion  is  influenced  greatly 
by  the  diet,  particularly  by  the  ingestion  of  fluids. 

Certain  pathological  conditions  cause  the  output  of  urine  for  any 
definite  period  to  depart  very  decidedly  from  the  normal  output. 
Among  the  pathological  conditions  in  which  the  volume  of  urine  is 
increased  above  normal  are  the  following:  Diabetes  mcllitus,  diabetes 
insipidus,  certain  diseases  of  the  nervous  system,  contracted  kidney, 
amyloid  degeneration  of  the  kidney,  and  in  convalescence  from  acute 
diseases  in  general.  Many  drugs  such  as  calomel,  digitalis,  acetates, 
and  salicylates  also  increase  the  volume  of  the  urine  excreted.  A 
decrease  from  the  normal  is  observed  in  the  following  pathological 
conditions:  Acute  nephritis,  diseases  of  the  heart  and  lungs,  fevers, 
diarrhcjea,  and  vomiting. 

Color. — Normal  urine  ordinarily  possesses  a  yellow  lint,  the  depth 
of  the  color  being  dependent  in  part  upon  the  density  of  the  fluid. 
The  color  of  normal  urine  is  due  principally  to  a  pigment  called  uro- 
chrome:  traces  of  lia-matoporp/iyrin,  urobilin,  and  urocrythrin  ha\'e  also 
been  detected.  Under  pathological  conditions  Ihc  urine  is  subject  to 
pronounccfl  variations  in  color  and  may  contain  man\-  varieties  of 
pigments.  Under  such  circumstances  the  urine  may  vary  in  color 
from  an  extremely  light  yellow  to  a  very  dark  brown  or  black.  Vogel 
has  constructed  a  color  chart  which  is  of  some  value  for  purposes  of 
comparison.  The  nature  and  origin  of  the  chief  variations  in  the 
urinary  color  arc  set  forth  in  tabular  form  by  Halliburton  as  follows: 

250 


URINE.  2:^1 


Color. 


Cause  of  Coloration. 


Pathological  Condition. 


Nearly  colorless i  Dilution,  o)»  diminution  of        Nervous  conditions:  h  y- 

normal  pigments.  druria,    diabetes    insipidus 


granular  kidnev. 


Dark   yellow  to    brown-red    Increase   of  normal,   or  oc-    Acute  febrile  diseases. 

currence     of     pathological, 
pigments. 


Milky Fat  globules Chyluria. 


Pus  corpuscles    Purulent     diseases     of     the 

urinar}'  tract. 

Orange    Excreted  drugs   ■   Santonin,  chrysophanic  acid. 

Red  or  reddish   ffematoporphyrin    Ha:morrhages,    or    h ae m o - 

Unchanged  haemoglobin.  .  . .      globinuria. 


Pigments  in  food  (logwood, 
madder,  bilberries,  fuchsin). 


Brown  to  brown-black 


Hsematin 

Small  haemorrhages. 

Methaemoglobin    

Methaemoglobinuria. 

Melanin 

Melanotic  sarcoma. 

Hydrochinon  and  catechol,  .i  Carbolic-acid  poisoning. 


Greenish-yellow,      greenish- 
brown,  approaching  black. 


Bile-pigments    Jaundice. 


Dirty  green '  or  blue 


A   dark-blue   scum   on   sur-  Cholera,  typhus;  seen  espe- 

face,    with   a  blue  deposit,  daily    when    the    urine    is 

due  to  an  excess  of  indigo-  putrefying, 
forming  substances. 


Brown-yellow  to  red-brown,  i  Substances  contained  in 
becoming  blood-red  upon  senna,  rhubarb,  and  che- 
adding  alkalis.  lidonium   which   are  intro- 

duced into  the  system. 


Transparency. — Normal  urine  is  ordinarily  perfectly  clear  and 
transparent  when  voided.  On  standing  for  a  variable  time,  however, 
a  cloud  (nubecula)  consisting  principally  of  nucleoprotein  or  mucoid 
(see  p.  284)  and  epithelial  cells  forms.  A  turbidity  due  to  the  pre- 
cipitation of  phosphates  is  normally  noted  in  urine  passed  after  a 
hearty  meal.  The  urine  obtained  2-3  hours  after  a  meal  or  later  is 
ordinarily  free  from  turbidity.  Permanently  turbid  urines  ordinarily 
arise  from  pathological  conditions. 

^  This  dirty  green  or  blue  color  also  occurs  after  the  use  of  methylene  blue  in  the 
organism. 


2=^2  PHYSIOLOGICAL    CHEMISTRY. 

Odor. — The  odor  of  normal  urine  is  of  a  faint,  aromatic  type. 
The  bodies  to  which  this  odor  is  due  are  not  well  known,  but  it  is 
claimed  bv  some  investisiators  to  be  due,  at  least  in  part,  to  the  pres- 
ence  of  minute  amounts  of  certain  volatile  organic  acids.  When  the 
urine  undergoes  decomposition,  e.  g.,  in  alkaline  fermentation,  a  very 
unpleasant  ammoniacal  odor  is  evolved.  All  urines  are  subject  to 
such  decomposition  if  allowed  to  stand  for  a  sufficiently  long  time. 
Under  normal  conditions  the  urine  very  often  possesses  a  peculiar 
odor  due  to  the  ingestion  of  some  certain  drug  or  ^•egetable.  For 
instance,  cubebs,  copaiba,  myrtol,  saffron,  tolu,  and  turpentine  each 
imparts  a  somewhat  specific  odor  to  the  urine.  After  the  ingestion  of 
asparagus,  the  urine  also  possesses  a  typical  odor. 

Frequency  of  Urination. — The  frequency  of  urination  \aries 
greatly  in  different  individuals  but  in  general  is  dependent  upon  the 
amount  of  fluid  in  the  bladder.  In  pathological  conditions  an  inflam- 
matory affection  of  the  urinary  tract  or  any  disturbance  of  the  inner\-a- 
tion  of  the  bladder  will  influence  the  frequency.  Affections  of  the 
spinal  cord  which  lead  to  an  increased  irritability  of  the  bladder  or  a 
weakening  of  the  sphincter  will  result  in  increasing  the  frecjuency  of 
urination. 

Reaction. — The  mixed  twenty-four  hour  urinary  excretion  of  a 
normal  individual  ordinarily  possesses  an  acid  reaction  to  litmus. 
This  acidity  is  now  believed  to  be  due  to  the  presence  of  various  acidic 
radicals  and  not  to  the  presence  of  sodium  di-hydrogen  phosphate  as 
was  formerly  held  (see  Phosphates,  p.  294).  This  conclusion  is 
reinforced  by  the  observation  that  urine  may  Ijc  divided  into  two 
portions,  one  part  consisting  almost  entirely  of  inorganic  matter, 
including  practically  (ill  of  the  phosphates  and  ha\ing  an  alkaline  reaction, 
the  other  containing  practically  all  of  the  organic  substances  and  no 
phosphates  and  having  an  acid  reaction.  The  acidity  imparted  to  the 
urine  by  any  particular  acid  depends  entirely  uyjon  the  extent  to 
which  the  acid  is  dissociable,  since  it  is  the  hydrogen  ion  which  is 
responsible  for  the  acid  reaction. 

The  comjiosition  of  the  food  is  jK'rhaps  the  most  imi)orlanl  factor 
in  determining  the  reaction  oi  the  urine.  The  reaction  ordinarily 
varies  considerably  according  to  the  time  of  day  the  urine  is  ])assed. 
Yor  instance,  for  a  variable  length  of  time  after  a  meal  the  urine  may 
be  neutral  or  even  alkaline  in  reaction  to  litmus,  owing  to  the  claim  of  the 
gastric  juice  ujjon  the  a(  idic  radicals  to  further  the  formation  of  hydro- 
chloric acid  for  use  in  carrying  out  the  digestive  secretory  function. 
This  change  in  reaction  is  known  as  the  alkaline  tide  anrl  is  common  to 


URINE. 


perfectly  healthy  individuals.  The  urine  may  also  become  temporarily- 
alkaline  in  reaction  to  litmus,  as  the  result  of  ingesting  alkaline  car- 
bonates or  certain  salts  of  tartaric  and  citric  acids  which  may  be  trans- 


FiG.  8i. — Deposit  in  Ammoniacal  Fermentation. 
a,  Acid  ammonium  urate;  b,  ammonium  magnesium  phosphate;  c,  bacteria. 

formed  into  carbonates  within  the  organism.  Normal  urine  upon 
standing  for  some  time  becomes  alkaline  in  reaction  to  litmus,  owing 
to  the  inception  of  alkaline  or  ammoniacal  fermentation  through  the 
agency  of  micro-organisms.  This  fermentation  has  no  especial  diag- 
nostic value  except  in  cases  where  the  urine  has  undergone  this  change 


'^k% 


^Af4  ,i% 


r^if 


o,i. 


'%%.    ^^rM'"^^ 


^^' 


^*  V 


Fig.  82. — Deposit  in  Acid  Fermentation. 
a,  Fungus,  b,  amorphous  sodium  urate;  c,  uric  acid;  d.  calcium  oxalate. 

within  the  organism  and  is  voided  in  the  decomposed  state.  iVmmoni- 
acal  fermentation  is  ordinarily  due  to  cystitis  or  occurs  as  the  result  of 
infection  in  the  process  of  catheterization.     A  microscopical  examina- 


'54 


PHYSIOLOGICAL    CHEMISTRY. 


tion  of  such  urine  (Fig.  8i,  p.  2>,;^)  shows  the  presence  of  ammonium 
magnesium  phosphate  crystals,  amorphous  phosphates,  and  not  infre- 
quently ammonium  urate. 

Occasionally  a  urine  which  possesses  a  normal  acidity  when 
voided,  upon  standing  instead  of  undergoing  ammoniacal  fermenta- 
tion as  above  described  will  become  still  more  strongly  acid  in  reaction. 
Such  a  phenomenon  is  termed  acid  fermentation. 
Accompanying  this  increased  acidity  there  is  ordinarily 
a  deepening  of  the  tint  of  the  urinary  color.  Such 
urines  may  contain  acid  urates,  uric  acid,  fungi,  and 
calcium  oxalate  (Fig.  82,  p.  253).  On  standing  for 
a  sufficiently  long  time  any  urine  which  exhibits  acid 
fermentation  will  ultimately  change  in  reaction,  due 
to  the  inception  of  alkaline  fermentation,  and  will 
show  the  microscopical  deposits  characteristic  of  such 
a  urine. 

Specific  Gravity. — The  specific  gravity  of  the 
urine  of  normal  individuals  varies  ordinarily  between 
1. 015  and  1.025.  This  value  is  subject  to  wide  fluc- 
tuations under  various  conditions.  For  instance, 
following  copious  water-  or  beer-drinking  the  specific 
gravity  may  fall  to  1.003  or  lower,  whereas  in  cases 
of  excessive  perspiration  it  may  rise  as  high  as  1.040 
or  e\en  higher.  Where  a  very  accurate  determination 
of  the  specific  gravity  is  desired  use  is  commonly  made 
of  the  pyknometer  or  of  the  Westphal  hydrostatic 
balance.  These  instruments,  however,  arc  not  suited 
for  clinical  use.  The  clinical  method  of  determining 
Fig.  8.i.~Uri.\-  the  specific  graxity  is  by  means  of  a  urinometer 
ivDER.  (Fig.  83,).    This  alTords  a  very  rapid  method  and  at 

the  same  time  is  sufficiently  accurate  for  clinical 
pLirpcjses.  The  urinometer  is  always  calibrated  for  use  at  a  specific 
temperature  and  the  observations  made  at  any  other  temperature 
must  be  subjected  t(;  a  certain  cc^rrection  to  obtain  the  true  specific 
gravity.  In  making  this  correction  one  unit  of  the  last  order  is  added 
to  the  observed  specific  gravity  for  every  three  degrees  above  the 
normal  temperature  and  subtracted  for  every  three  degrees  below 
the  normal  temperature.  For  instance,  if  in  using  a  urinometer 
calibrated  for  15°  C.  the  specific  gravity  of  a  urine  having  a 
temperature  of  21°  C  is  determined  as  r.oi8  it  is  necessary  to  add  to 
the  observed  specific  gravity  two  units  of  the  third  order  to  obtain 


,< 


URINE.  255 

the  real  specific  gravity  of  the  urine.  Therefore  the  true  specific 
gravity,  at  15°  C,  of  a  urine  having  a  specific  gravity  of  1.018  at  21° 
C.  is  1.018  +  0.002  =  1.020. 

Pathologically,  the  specific  gravity  may  be  subjected  to  very  wide 
variations.  This  is  especially  true  in  diseases  of  the  kidneys.  In  acute 
nephritis  ordinarily  the  urine  is  concentrated  and  of  a  high  specific 
gravity,  whereas  in  chronic  nephritis  the  reverse  conditions  are  more 
apt  to  prevail.  In  fact,  under  most  conditions,  whether  physiological 
or  pathological,  the  specific  gravity  of  the  urine  is  inversely  propor- 
tional to  the  volume  excreted.  This  is  not  true  of  diabetes  mellitus, 
however,  where  the  volume  of  urine  is  large  and  the  specific  gravity  is 
also  high,  owing  to  the  sugar  contained  in  the  urine. 

The  amount  of  solids  eliminated  in  the  excretion  for  twenty-four 
hours  may  be  roughly  calculated  by  means  oiLong's  coefficient,  i.  e.,  2.6. 
The  solid  content  of  1000  c.c.  of  urine  is  obtained  by  multiplying  the 
last  two  figures  of  the  specific  gravity  observed  at  25°  C.  by  2.6.  To 
determine  the  amount  of  solids  excreted  in  twenty-four  hours  if  the 
volume  was  1120  c.c.  and  the  specific  gravity  was  1.018  the  calcula- 
tion would  be  as  follows: 

(a)   18  X  2.6  =  46.8  grams  of  solid  matter  in  1000  c.c.  of  urine. 

(h)   4_j — ^  ^-'-^^  =  c;2.4  grams  of  sohd  matter  in  11 20  c.c.  of  urine. 
^  1000  J    -^  G 

The  coefficient  of  Haser  (2.33)  which  has  been  in  use  for  years 
probably  gives  values  that  are  inaccurate  for  conditions  existing  in 
America.  This  coefficient  was  calculated  on  the  basis  of  the  specific 
gravity  determined  at  a  temperature  of  15°  C. 

Freezing-point  (Cryoscopy). — The  freezing-point  of  a  solution 
depends  upon  the  total  number  of  molecules  of  solid  matter  dissolved 
in  it.  The  determination  of  the  osmotic  pressure  by  this  method  has 
recently  come  to  be  of  some  clinical  importance,  particularly  as  an  aid 
in  the  diagnosis  of  kidney  disorders.  In  this  connection  it  is  best  to 
collect  the  urine  from  each  kidney  separately  and  determine  the  freez- 
ing-point in  the  individual  samples  so  collected.  By  this  means  con- 
siderable aid  in  the  diagnosis  of  renal  diseases  may  be  secured.  The 
fluids  most  frequently  examined  cryoscopically  are  the  blood  (see  p. 
178)  and  the  urine.  The  freezing-point  is  denoted  by  A.  The  value 
of  A  for  normal  urine  varies  ordinarily  between  ■ — 1.3°  and — 2.3°  C, 
the  freezing-point  of  pure  water  being  taken  as  0°.  A  is  subject  to 
very  wide  fluctuations  under  unusual  conditions.  For  instance,  fol- 
lowing copious  water-  or  beer-drinking  A  may  have  as  high  a  value  as 
— 0.2°  C,  whereas  on  a  diet  containing  much  salt  and  deficient  in  fluids 


2^6 


PHYSIOLOGICAL    CHEMISTRY. 


'n 


the  value  of  Amav  be  lowered  to  — 3°  C.  or  e\en  lower.  The  freezing- 
point  of  normal  blood  is  generally  about  — 0.56°  C.  and  is  not  subject 
to  the  wide  variations  noted  in  the  urine, 
because  of  the  tendency  of  the  organism  to 
maintain  the  normal  osmotic  pressure  of  the 
blood  under  all  conditions.  Variations  be- 
tween — 0.51°  and  — 0.62°  C.  may  be  due 
entirely  to  dietary  conditions,  but  if  any 
marked  variation  is  noted  it  can,  in  most 
cases,  be  traced  to  a  disordered  kidney 
function. 

Freezing-point  determinations  may  be 
made  by  means  of  the  Beckmann-Heidenhain 
apparatus  (Fig.  84)  or  the  Zikel  pektoscope. 
The  Beckmann-Heidenhain  apparatus  con- 
sists of  the  following  parts:  A  strong  battery 
jar  or  beaker  (C)  furnished  with  a  metal  cover 
which  is  provided  with  a  circular  hole  in  its 
center.  This  strong  glass  vessel  serves  to 
hold  the  freezing  mixture  by  means  of  which 
the  temperature  of  the  fluid  under  examination 
is  lowered.  A  large  glass  tube  (B)  designed  as 
an  air-jacket,  and  formed  after  the  manner 
of  a  test-tube  is  introduced  through  the 
central  aperture  in  the  metal  cover  and  into 
this  air- jacket  is  lowered  a  smaller  tube  (A) 
containing  the  fluid  to  lie  tested.  A  very 
delicate  thermometer  (D),  graduated  in 
liundredths  of  a  degree  is  introduced  into  the 
inner  tube  and  is  held  in  ])lace  by  means  of  a 
cork  so  th;it  the  mercury  !)ull)  is  ininirrsed  in 
the  fluid  under  examination  but  does  not  come 


Fig.  S4.— BECK.\tANN- 
H  e  I  d  e  -v  h  a  I  n  Freezino- 
I'oiNT  .Apparatus.     {Long.) 

1),  a  delicate  thermom- 
eter; C,  the  containing  jar; 

/f,  the  outside  or  air  mantle      j,-,  contact  with  any  glass  surface.      A  small 

tube;  A,  the  tube  in  which  .  .  .  i         i    •  i 

]>l;itnuim  wire  stirrer  serves  to  keep  the  lluid 

under  examination   we 


the  mixture  to  be  (observed 
is  j^laced.  'I\v<j  stirrers 
are  shown,  one  for  the 
cofjling  mixture  in  the  jar 
and  one  for  the  experi- 
mental mixture. 


mixed   Willie  a  larger 

stirrer    is    used    to    mani|)iil;ttc    I  he    freezing 

mixture.      (Rock  salt  and  ice  in  the  (troportion 

1  :3  form  a  very  satisfactory  freezing  mixture.) 

In  making  a  determinati(;n  of  the  freezing-j>oint  of  a  fluid  b_\'  means 

of  the  Heckmann  Ilcidenhain  ap|)aratLis  proceed  as  follows:     IMace  the 

freezing  mixture  in  ihe  batter}'  j.'ir.ind  ;idd  \v;iler  (if  necessary)  to  secure 


URINE.  257 

a  temperature  not  lower  than  3°  C.  Introduce  the  fluid  to  be  tested 
into  tube  A,  place  the  thermometer  and  platinum  wire  stirrer  in  posi- 
tion, and  insert  the  tube  into  the  air-jacket  which  has  previously  been 
inserted  through  the  metal  cover  of  the  battery  jar.  Manipulate  the 
two  stirrers  in  order  to  insure  an  equalization  of  temperature  and 
observe  the  course  of  the  mercury  column  of  the  thermometer  very 
carefully.  The  mercury  will  gradually  fall  and  this  gradual  lowering 
of  the  temperature  will  be  followed  by  a  sudden  rise.  The  point  at 
which  the  mercury  rests  after  this  sudden  rise  is  the  freezing-point. 
This  rise  is  due  to  the  fact  that  previous  to  freezing,  a  fluid  is  always 
more  or  less  overcooled  and  the  thermometer  temporarily  registers  a 
temperature  somewhat  below  the  freezing-point.  As  the  fluid  freezes, 
however,  there  is  a  very  sudden  change  in  the  temperature  of  the  liquid 
and  this  change  is  imparted  to  the  thermometer  and  causes  the  rise  as 
indicated.  It  occasionally  occurs  that  the  fluid  under  examination  is 
very  much  overcooled  and  does  not  freeze.  Under  such  circumstances 
a  small  piece  of  ice  is  introduced  into  it  by  means  of  the  side  tube  noted 
in  the  figure.  This  so-called  "inoculation"  causes  the  fluid  to  freeze 
instantaneously.  (For  details  of  the  method  of  determining  the  freez- 
ing-point consult  standard  works  on  physical  or  organic  chemistry.) 

Electrical  Conductivity. — The  electrical  conductivity  of  the 
urine  is  dependent  upon  the  number  of  inorganic  molecules  or  ions 
present,  and  in  this  differs  from  the  freezing-point  which  is  dependent 
upon  the  total  number  of  molecules  both  inorganic  and  organic  which 
are  in  solution.  The  conductivity  of  the  urine  has  been  investigated 
but  slightly,  and  this  very  recently,  but  from  the  data  secured  it  seems 
that  the  value  generally  falls  below  k  =  0.03.  The  conductivity  of  blood 
serum  has  been  determined  as  k  =  0.012.  Up  to  the  present  time  the 
determination  of  the  electrical  conductivity  of  any  of  the  fluids  of  the 
body  has  been  put  to  very  slight  clinical  use.  Experience  may  show 
the  conductivity  value  to  be  a  more  important  aid  to  diagnosis  than  it 
is  now  considered,  particularly  if  it  is  taken  in  connection  with  the 
determination  of  the  freezing-point.  By  a  combination  of  these  two 
methods  the  portion  of  the  osmotic  pressure  due  respectively  to 
electrolytes  and  non-electrolytes  may  be  determined.  For  a  discussion 
of  electrical  conductivity,  the  method  by  which  it  is  determined,  and 
the  principles  involved  consult  standard  works  on  physical  or  electro- 
chemistry. 

Collection  of  the  Urine  Sample. — If  any  dependable  data  are 
desired  regarding  the  quantitative  composition  of  the  urine  the  examina- 
tion of  the  mixed  excretion  for  twenty-four  hours  is  absolutely  necessary. 
17 


PHYSIOLOGICAL   CHEMISTRY. 


In  collecting  the  urine  the  bladder  may  be  emptied  at  a  given  hour, 
say  8  A.  M.,  the  urine  discarded  and  all  the  urine  from  that  hour  up 
to  and  including  that  passed  the  next  day  at  8  A.  M.,  saved,  thoroughly 
mixed,  and  a  sample  taken  for  analysis.     Powdered  thymol. 


OH 

CH.j  —  CH  —  CHg, 

is  a  very  satisfactory  preservative  since  the  excess  may  be  remo^'ed  by 
filtration,  if  desired,  and  any  small  amount  which  may  go  into  solution 
will  have  no  appreciable  influence  upon  the  determination  of  any  of 
the  urinary  constituents.  It  has  no  reducing  power  and  so  may  safely 
be  used  to  preserve  diabetic  urines.  To  insure  the  preservation  of  the 
mixed  urine  of  the  twenty-four  hour  period  it  is  advisable  to  place  a 
small  amount  of  the  thymol  powder  in  the  urine  receptacle  before  the 
first  fraction  of  urine  is  voided.  In  order  to  further  insure  the  preser- 
vation of  the  urine  the  cleaned  and  dried  urine  receptacle  may  be  rinsed 
with  an  alcoholic  solution  of  thymol  and  subsequently  thoroughly  dried 
before  introducing  the  urine. 

Toluene  is  also  used  for  the  preser\'ation  of  urine. 

In  certain  pathological  conditions  it  is  desiraljle  to  collect  the  urine 
passed  during  the  day  separately  from  that  ])assed  during  the  night. 
When  this  is  done  the  urine  voided  between  8  A.  M.  and  8  P.  M.  may 
be  taken  as  the  day  sample  and  that  voided  between  8  P.  M.  and  8 
A.  M.  as  the  night  sample. 

The  qualitative  testing  of  urine  voided  at  random,  except  in  a  few 
specific  instances,  is  of  no  particular  value  so  far  as  giving  us  any 
accurate  knowledge  as  to  the  exact  urinary  characteristics  of  the  indi- 
vidual is  concerned.  In  the  great  majority  of  cases  the  qualitative  as 
well  as  the  quantitative  tests  should  be  made  iijjon  the  mixed  excretion 
for  a  twenty  four  hour  period. 


CHAPTER  XVIII. 


URINE:  PHYSIOLOGICAL  CONSTITUENTS. 
I.  Organic  Physiological  Constituents. 


Urea. 
Uric  acid. 
Creatinine. 
Creatine. 

Ethereal  sulphuric  acids. 

Hippuric  acicl. 
Oxalic  acid. 


Indoxyl-sulphuric  acid. 
Phenol-  and  ^-cresol-sulphuric  acids. 
Pyrocatechin-sulphuric  acid. 
Skatoxyl-sulphuric  acid. 


Neutral  sulphur  compounds. 


Allantoin. 


Aromatic  oxvacids . 


Cystine. 

Chondroitin-sulphuric  acid. 
Thiocyanates. 
Taurine  derivatives. 
Oxyproteic  acid. 
Alloxyproteic  acid. 
Uroferric  acid. 

Paraoxyphenyl-acetic  acid. 

Paraoxyphenyl-propionic  acid. 

Homogentisic  acid. 

Uroleucic  acid. 

Oxymandelic  acid. 

Kynurenic  acid. 
Benzoic  acid. 
Neucleoprotein. 
Oxaluric  acid. 

'It  is  impossible  to  make  any  absolute  classification  of  the  physiological  and  path- 
ological constituents  of  the  urine.  A  substance  may  be  present  in  the  uiine  in  small 
amount  physiologically  and  be  sufbciently  increased  under  certain  conditions  as  to  be 
termed  a  pathological  constituent.  Therefore  it  depends,  in  some  instances,  upon  the 
quantity  of  a  constituent  present  whether  it  may  be  correctly  termed  a  physiological  or  a 
pathological  constituent. 


2  bo 


PHYSIOLOGICAL    CHEMISTRY, 


Phosphorized  compounds. 


[  Pepsin. 
Enzymes   |  Gastric  rennin. 

[  Amylase. 

[  Acetic  acid. 
\'olatile  fatty  acids I  Butyric  acid. 

1^  Formic  acid. 
Paralactic  acid. 
Phcnaceturic  acid. 

Glycerophosphoric  acid. 
Phosphocarnic  acid. 

[  Urochrome. 
Pigments ' Uroblin. 

[  Uroerythrin. 

Adenine. 

Guanine. 

Xanthine. 

Epiguanine. 

Episarkine. 

Hypoxanthine. 

Paraxanthine. 

Heteroxanthine. 

i-Methylxanthinc. 

2.  Inorganic  Physiological  Constituents. 


Ptomaines  and  leucomaines. 


Purine  liases 


.\mmonia. 

Sulphates. 

Chlorides. 

Phosphates. 

Sodium  and  potassium. 

Calcium  and  magnesium. 

Carbonates. 

lr(jn. 

Fluorides. 

-Xitrates. 

Silicates. 

Hydrogen  jjeroxide. 


NH., 

I 
UREA,  C  =  (). 

I 

NIL. 


URINE. 


261 


Urea  is  the  principal  end-product  of  the  metabolism  of  protein 
substances.  It  has  been  generally  believed  that  about  90  per  cent 
of  the  total  nitrogen  of  the  urine  was  present  as  urea.  Recently, 
however,  Folin  has  shown  that  the  distribution  of  the  nitrogen  of  the 
urine  among  urea  and  the  other  nitrogen-containing  bodies  present 
depends  entirely  upon  the  absolute  amount  of  the  total  nitrogen 
excreted.  He  found  that  a  decrease  in  the  total  nitrogen  excretion 
was  always  accompanied  by  a  decrease  in  the  percentage  of  the  total 
nitrogen  excreted  as  urea,  and  that  after  so  regulating  the  diet  of  a 


Fig.  85. — Urea. 

normal  person  as  to  cause  the  excretion  of  total  nitrogen  to  be  reduced 
to  3-4  grams  in  24  hours,  only  about  60  per  cent  of  this  nitrogen  appeared 
in  the  urine  as  urea.  His  experiments  also  seem  to  show  urea  to  be  the 
only  one  of  the  nitrogenous  excretions  which  is  relatively  as  well  as 
absolutely  decreased  as  a  result  of  decreasing  the  amount  of  protein 
metabolized.  This  same  investigator  reports  a  hospital  case  in  which 
only  14.7  per  cent  of  the  total  nitrogen  was  present  as  urea  and  about 
40  per  cent  was  present  as  ammonia.  Morner  had  previously  reported 
a  case  in  which  but  4.4  per  cent  of  the  total  nitrogen  of  the  urine  was 
present  as  urea,  and  26.7  per  cent  was  present  as  ammonia. 
(  Urea  occurs  most  abundantly  in  the  urine  of  man  and  carnivora 
and  in  somewhat  smaller  amount  in  the  urine  of  herbivora;  the  urine 
of  fishes,  amphibians,  and  certain  birds  also  contain  a  small  amount  of 
the  substance.  Urea  is  also  found  in  nearly  all  the  fluids  and  in  many 
of  the  tissues  and  organs  of  mammals.  The  amount  excreted,  under 
normal  conditions,  by  an  adult  man  in  24  hours  is  about  30  grams; 
women  excrete  a  somewhat  smaller  amount.     The  excretion  is  greatest 


262  PHYSIOLOGICAL    CHEMISTRY. 

in  amount  after  a  diet  of  meat,  and  least  in  amount  after  a  diet  con- 
sisting of  non-nitrogenous  foods;  this  is  due  to  the  fact  that  the  last- 
mentioned  diet  has  a  tendency  to  decrease  the  metabolism  of  the  tissue 
proteins  and  thus  cause  the  output  of  urea  under  these  conditions  to 
fall  below  the  output  of  urea  observed  during  starvation.  The  output 
of  urea  is  also  increased  after  copious  water-  or  beer-drinking.  The 
increase  is  probably  due  primarily  to  the  washing  out  of  the  tissues  of 
the  urea  previously  formed,  but  which  had  not  been  removed  in  the 
normal  processes,  and  secondarily  to  a  stimulation  of  protein  catabolism. 
Urea  may  be  formed  in  the  organism  from  amino  acids  such 
as  leucine,  glycocoU,  and  aspartic  acid:  it  may  also  be  formed 
from  ammonium  carbonate  (NHJ2CO3  or  ammonium  carbamate, 
H.N.O.CO.NH,. 

There  are  differences  of  opinion  regarding  the  transformation  of 
the  substances  just  named  into  urea,  but  there  is  rather  conclusive 
evidence  that  at  least  a  part  of  the  urea  is  formed  in  the  liver;  it  may 
be  formed  in  other  organs  or  tissues  as  well. 

Urea  crystallizes  in  long,  colorless,  four-  or  six-sided,  anhydrous,  rhom- 
bic prisms  (Fig.  85,  p.  261),  which  melt  at  132°  C.  and  are  soluble  in 
water  or  alcohol  and  insoluble  in  ether  or  chloroform.  If  a  crystal 
of  urea  is  heated  in  a  test-tube,  it  melts  and  decomposes  with  the  libera- 
tion of  ammonia.     The  residue  contains  cyanuric  acid, 

COH 

/\ 

N       N 

HO.C       COH 

\/ 

N 

and  hiurel, 

NH2 

1 

c=o 

\ 
NH 

/ 

c=o 

I 
NH, 

The  biuret  may  be  dissolved  in  water  and  a  reddish-violet  color 
obtained  by  treating  the  aqueous  solution  with  cu])ric  sulj)hate  and 
potassium  hydroxide  (see  iiiuret  Test,  p.  90J.     Uerlain  hypochlorites 


URINE. 


26' 


or  hypobromites  in  alkaline  solution  have  the  power  of  decomposing 
urea  into  nitrogen,  carbon  dioxide,  and  water.  Sodium  hypobromite 
brings  about  this  decomposition,  as  follows: 

CO(NH2)2  +  3NaOBr  =  3NaBr  +  N2  +  C02  +  2H20. 

This  property  forms  the  basis  for  a  clinical  quantitative  determination 
of  urea  (see  page  369). 

Urea  has  the  power  of  forming  crystalline  compounds  with  certain 
acids;  urea  nitrate  and  urea  oxalate  are  the  most  important  of  these 
compounds.  Urea  nitrate,  CO(NH2)2-HN03,  crystallizes  in  colorless, 
rhombic  or  six-sided  tiles  (Fig.  86,  below),  which  are  easily  soluble  in 
water.  Urea  oxalate,  2.CO(NH2)2.H2C204,  crystallizes  in  the  form 
of  rhombic  or  six-sided  prisms  or  plates  (Fig.  88,  p.  265) :  the  oxalate 
differs  from  the  nitrate  in  being  somewhat  less  soluble  in  water. 


Fig. 


-Urea  Nitr.a.te. 


A  decrease  in  the  excretion  of  urea  is  observed  in  many  diseases  in 
which  the  diet  is  much  reduced  and  in  some  disorders  as  a  result  of 
alterations  in  metabolism,  e.  g.,  myxoedema,  and  in  others  as  a  result 
of  changes  in  excretion,  as  in  severe  and  advanced  kidney  disease.  A 
pathological  increase  is  found  in  a  large  proportion  of  diseases  which 
are  associated  with  a  toxic  state. 


Experiments  on  Urea. 

I.  Isolation  from  the  Urine. — Place  800  c.c.  of  urine  in  a  pre- 
cipitating jar,  add  250  c.c.   of  baryta  mixture,^  and  stir  thoroughly. 

^  Barvta  mixture  consists  of  a  mixture  of  one  volume  of  a   saturated   solution  of 
Ba(N03^2  and  two  volumes  of  a  saturated  soiution  of  Ba(OH)3. 


^64 


PHYSIOLOGICAL    CHEMISTRY. 


Filter,  off  the  precipitate  of  phosphates,  sulphates,  urates,  and  hippu- 
rates  and  evaporate  the  filtrate  on  a  water-bath  to  a  thick  syrup.  This 
syrup  contains  chlorides,  creatinine,  organic  salts,  pigments,  and  urea. 
Extract  the  svrup  with  warm  95  per  cent  alcohol  and  filter  again.  The 
filtrate  contains  the  urea  contaminated  with  pigment.  Decolorize  the 
filtrate  bv  boiling  with  animal  charcoal,   filter  again,  and  stand  the 

filtrate  away  in  a  cold  place  for  crystallization. 

Examine  the  crystals  under  the  microscope  and* 

compare    them    with    those    shown    in    Fig.   85, 

page  261. 

2.  Solubility. — Test  the  solubility  of  urea, 
prepared  by  yourself  or  furnished  by  the  in- 
structor, in  the  ordinary  solvents  (see  p.  22)  and 
in  alcohol  and  ether. 

3.  Melting-point. — Determine  the  melting- 
point  of  some  pure  urea  furnished  by  the  in- 
structor. Proceed  as  follows:  Into  an  ordinary 
melting-point  tube,  scaled  at  one  end,  introduce 
a  crystal  of  urea.  Fasten  the  tube  to  the  bulb  of 
a  thermometer  as  shown  in  Fig.  87,  and  suspend 
the  bulb  and  its  attached  tube  in  a  small  beaker 
containing  sulphuric  acid.  Gently  raise  the 
temperature  of  the  acid  by  means  of  a  low  flame, 
stirring  the  fluid  continually,  and  note  the  tem- 
j^erature  at  which  the  urea  begins  to  melt. 

4.  Crystalline  Form. — Dissolve  a  crystal  of 
pure  urea  in  a  few  drops  of  0=:  per  cent  alcohol 

I'lG.     87.— -Meltixg-       '  .  . 

POINT  Tubes  Fastened     and  place  1-2  drops  of  the  alcoholic  solution  on 

TO    Bulb    of    The r-  .  .       ,.  ,  .,,         ,,         1      u    1  4         

.MO.METER  ^  microscopic  slide.     Allow  the  alcohol  to  evap- 

orate spontaneously,  examine  the  crystals  under 
the  microscope,  and  compare  them  with  those  re]>r()(luce(l  in  Fig.  85, 
p.  261.  Recrystallize  a  little  urea  from  water  in  the  same  way  and 
compare  the  crystals  with  those  obtained  from  the  alcoholic  solution. 

5.  Formation  of  Biuret. — Place  a  small  amount  of  urea  in  a  dry 
test-tube  and  heat  carefully  in  a  low  flame.  The  urea  melts  at  132°  C. 
and  liberates  ammonia.  Continue  heating  until  the  fused  mass  begins 
to  solidify.  Cool  the  tube,  dissolve  the  residue  in  dilule  ])Otassium 
hydroxide  solution,  and  add  very  dilute  cuj^ric  suljjhate  solulion  (see  p. 
90J.  The  purplish-violet  color  is  due  to  the  presence  of  biuret  which 
has  been  formed  from  the  urea  through  the  application  of  heat  as 
indicated.     This  is  the  reaction: 


URINE. 

NH2 

2   C=0 

Urea. 

c=o 

\ 

NH+NH3 

/ 

c=o 

NH, 

26: 


Biuret. 


6.  Urea  Nitrate. — Prepare  a  concentrated  solution  of  urea  by 
dissolving  a  little  of  the  substance  in  a  few  drops  of  water.  Place  a 
drop  of  this  solution  on  a  microscopic  slide,  add  a  drop  of  concentrated 
nitric  acid,  and  examine  under  the  microscope.  Compare  the  crystals 
with  those  reproduced  in  Fig.  86,  p.  263. 

7.  Urea  Oxalate. — To  a  drop  of  a  concentrated  solution  of  urea, 
prepared  as  described  in  the  last  experiment  (6),  add  a  drop  of  a 


Fig. 


Urea  Ox.\late. 


saturated  solution  of  oxalic  acid.     Examine  under  the  microscope  and 
compare  the  crystals  with  those  shown  in  Fig.  88,  above. 

8.  Decomposition  by  Sodium  Hypobromite. — Into  a  mixture 
of  3  c.c.  of  concentrated  sodium  hydroxide  solution  and  2  c.c.  of  bro- 
mine water  in  a  test-tube  introduce  a  crystal  of  urea  or  a  small  amount 
of  concentrated  solution  of  urea.  Through  the  influence  of  the  sodium 
hypobromite,  NaOBr,  the  urea  is  decomposed  and  carbon  dioxide 
and  nitrogen  are  liberated.  The  carbon  dioxide  is  absorbed  by  the 
excess  of  sodium  hydroxide,  while  the  nitrogen  is  evolved  and  causes 


266  PHYSIOLOGICAL    CHEMISTRY. 

the  marked  effervescence  observed.     This  property  forms  the  basis 
for  one  of  the  methods  in  common  use  for  the  quantitative  determina-  ■ 
tion  of  urea.     Write  the  equation  showing  the  decomposition  of  urea 
by  sodium  hypobromite.  ; 

9.  Furfurol  Test.—  To  a  few  crystals  of  urea  in  a  small  porcelain 
dish  add  1-2  drops  of  a  concentrated  aqueous  solution  of  furfurol  and 
1-2  drops  of  a  concentrated  hydrochloric  acid.  Note  the  appearance 
of  a  yellow  color  which  gradually  changes  into  a  purple.  AUantoin 
also  responds  to  this  test  (see  page  280). 

HN-C=0 
URIC  ACID,   OC      C-NH 


^>C0. 
HN-C-NH 

Uric  acid  is  one  of  the  most  important  of  the  constituents  of  the 
urine.  It  is  generally  stated  that  normally  about  0.7  gram  is  excreted 
in  24  hours  but  that  this  amount  is  subject  to  wide  variations,  particu- 
larly under  certain  dietary  and  pathological  conditions.  Very  recently 
it  has  been  shown  that  the  average  daily  excretion  of  uric  acid  for  ten 
men  ranging  in  age  from  19  to  29  years  and  fed  a  normal  mixed  diet 
was  0.597  gram,  a  value  somewhat  lower  than  the  generally  accepted 
average  of  0.7  gram  for  such  a  period.  Uric  acid  is  a  diureidc  and 
consequently  upon  oxidation  yields  two  molecules  of  urea.  It  acts 
as  a  weak  dibasic  acid  and  forms  two  classes  of  salts,  neutral  and  acid. 
The  neutral  potassium  and  lithium  urates  are  the  most  easily  soluble 
of  the  alkali  salts;  the  ammonium  urate  is  diflicultly  soluble.  The 
acid-alkali  urates  are  more  insoluble  and  form  the  major  portion  of  the 
sediment  which  separates  upon  cooling  the  concentrated  urine;  the 
alkaline  earth  urates  are  \'ery  insoluble.  Ordinarily  uric  acid  occurs 
in  the  urine  in  the  form  of  urates  and  upon  acidifying  the  licjuid  the 
uric  acid  is  liberated  and  dei)osits  in  crystalline  form.  This  property 
forms  the  basis  of  one  of  the  older  methods  for  the  (|uantitative  deter- 
mination of  uric  acid  (Heintz  Method,  p.  367). 

Uric  acid  is  very  closely  related  to  the  purine  bases  as  may  be  seen 
from  a  com])arison  of  its  structural  formula  with  those  of  the  purine 
bases  given  on  page  237.  According  to  the  ])urine  nomenclature  it  is 
designated  2-6-8-trioxy|)iirinc.  Uric  acid  forms  I  he  principal  end- 
product  of  the  nitrogenous  metabolism  oi  birds  and  scaly  amphibians; 
in  the  human  organism  it  occujjies  the  fourth  ])osition  inasmuch  as  here 
urea,  ammonia,  and  creatinine  are  the  chief  end  products  of  nitrogenous 


PL  \TK  V. 


Vk\c  AfiD  Crystals.     N'okmm.  C'u.dK.     (Iioni  I'nrdy,  after  fryer.) 


URINE.  267 

metabolism.  It  is  generally  said  that  the  relation  existing  between 
uric  acid  and  urea  in  human  urine  under  normal  conditions  varies  on 
the  average  from  1 140  to  i :  100  and  is  subject  to  wider  variations  under 
pathological  conditions;  and  further  tlfat  because  of  the  high  content  of 
uric  acid  in  the  urine  of  new-born  infants  the  ratio  may  be  reduced  to 
1 :  10  or  even  lower.  We  now  know  that  this  ratio  of  uric  acid  to  urea 
is  of  little  significance  under  any  conditions. 

In  man,  uric  acid  probably  results  principally  from  the  destruction 
of  nuclein  material.  It  may  arise  from  nuclein  or  other  purine  material 
ingested  as  food  or  from  the  disintegrating  cellular  matter  of  the  organ- 
ism. The  uric  acid  resulting  from  the  first  process  is  said  to  be  of 
exogenous  origin,  whereas  the  product  of  the  second  form  of  activity  is 
said  to  be  of  endogenous  origin.  As  a  result  of  experimentation,  Siven. 
and  Burian  and  Schur,  and  Rockwood  claim  that  the  amount  of  endog- 
enous uric  acid  formed  in  any  given  period  is  fairly  constant  for  each 
individual  under  normal  conditions,  and  that  it  is  entirely  independent 
of  the  total  amount  of  nitrogen  eliminated.  Recently  Folin  has  taken 
exception  to  the  statements  of  these  investigators  and  claims  that, 
following  a  pronounced  decrease  in  the  amount  of  protein  metabolized, 
the  absolute  quantity  of  uric  acid  is  decreased  but  that  this  decrease  is 
relatively  smaller  than  the  decrease  in  the  total  nitrogen  excretion  and 
that  the  per  cent  of  the  uric  acid  nitrogen,  in  terms  of  the  total  nitrogen, 
is  therefore  decidedly  increased. 

In  birds  and  scaly  amphibians  the  formation  of  uric  acid  is  analo- 
gous to  the  formation  of  urea  in  man.  '  In  these  organisms  it  is  derived 
principally  from  the  protein  material  of  the  tissues  and  the  food  and  is 
formed  through  a  process  of  synthesis  which  occurs  for  the  most  part 
in  the  liver;  a  comparatively  small  fraction  of  the  total  uric  acid  excre- 
tion of  birds  and  scaly  amphibians  may  result  from  nuclein  material. 

When  pure,  uric  acid  may  be  obtained  as  a  white,  odorless,  and 
tasteless  powder,  which  is  composed  principally  of  small,  transparent, 
crystalline,  rhombic  plates.  Uric  acid  as  it  separates  from  the  urine 
is  invariably  pigmented,  and  crystallizes  in  a  large  variety  of  character- 
istic forms,  e.  g.,  dumb-bells,  wedges,  rhombic  prisms,  irregular  rect- 
angular or  hexagonal  plates,  whetstones,  prismatic  rosettes,  etc.  Uric 
acid  is  insoluble  in  alcohol  and  ether,  soluble  with  difficulty  in  boiling 
water  (1:1800)  and  practically  insoluble  in  cold  water  (1:39,480,  at 
18°  C).  It  is  soluble  in  alkalis,  alkali  carbonates,  boiling  glycerol, 
concentrated  sulphuric  acid,  and  in  certain  organic  bases  such  as  ethyl- 
amine  and  piperidine.  It  is  claimed  that  the  uric  acid  is  held  in  solu- 
tion in  the  urine  by  the  urea  and  disodium  hydrogen  phosphate  present. 


268  PHYSIOLOGICAL    CHEMISTRY. 

Uric  acid  possesses  the  power  of  reducing  cupric  hydroxide  in  alkaUne 
solution  and  may  thus  lead  to  an  erroneous  conclusion  in  testing  for 
sugar  in  the  urine  by  means  of  Fehling's  or  Trommer's  tests.  A  white 
precipitate  of  cuprous  urate  is  formed  if  only  a  small  amount  of  cupric 
hydroxide  is  present,  but  if  enough  of  the  copper  salt  is  present  the 
characteristic  red  or  brownish-red  precipitate  of  cuprous  oxide  is  ob- 
tained. Uric  acid  does  not  possess  the  power  of  reducing  bismuth  in 
alkaline  solution  and  therefore  does  not  interfere  in  testing  for  sugar  in 
the  urine  by  means  of  Boettger's  or  Nylander's  tests. 

In  addition  to  being  an  important  urinary  constituent  uric  acid  is 
normally  present  in  the  brain,  heart,  liver,  lungs,  pancreas,  and  spleen; 
it  also  occurs  in  the  blood  of  birds  and  has  been  detected  in  traces  in 
human  blood  under  normal  conditions. 

Pathologically,  the  excretion  of  uric  acid  is  subject  to  wide  variations, 
but  the  experimental  findings  are  rather  contradictory.  It  may  be 
stated  with  certainty,  however,  that  in  leukaemia  the  uric  acid  output 
is  increased  absolutely  as  well  as  relatively  to  the  urea  output;  under 
these  conditions  the  ratio  between  the  uric  acid  and  urea  may  be  as 
low  as  1:9,  whereas  the  normal  ratio,  as  we  have  seen,  is  i :  50  or  higher. 
In  the  study  of  the  intiuence  of  X-ray  on  metabolism  Edsall  has  very 
recently  reached  some  interesting  conclusions.  He  found  that  the 
excretion  of  uric  acid  is  usually  increased  and  that  in  some  conditions, 
particularly  in  leukaemia,  it  may  be  greatly  increased.  The  excretion 
of  total  nitrogen,  phosphates,  and  other  substances  may  also  l)c  con- 
siderably increased. 

1'>\i'i:rimi;.\t.s  ox  Uric  Acid. 

1.  Isolation  from  the  Urine.  — Place  about  200  c.c.  of  filtered 
urine  in  a  beaker,  render  it  acid  with  2-10  c.c.  of  concentrated  hydro- 
chloric acid,  stir  thoroughly,  and  stand  the  \essel  in  a  cold  place  for  24 
hours.  Ivxamine  the  jjigmented  crystals  of  uric  acid  under  the  micro- 
scope and  compare  them  with  those  shown  in  Fig.  101,  \).  342  and  PI. 
\'.,  opposite  p.  267. 

2.  Solubility. — Try  the  solubility  of  ])ure  uric  acid,  furnished  by 
the  instructor,  in  the  ordinary  solvents  (see  ]).  22)  and  in  alcohol,  ether, 
concentrated  sulphuric  acid  and  in  boiling  glycerol. 

3.  Crystalline  Form  of  Pure  Uric  Acid.  Phut  aJjoul  100  c.c.  of 
water  in  a  small  beaker,  render  it  distinctly  alkaline  with  ])otassium 
hydroxide  solution  and  add  a  small  amount  of  ])ure  uric  acid,  stirring 
continuously.     Cool  the  solution,  render  il  distinctly  acid  with  hydro- 


URINE.  269 

chloric  acid  and  allow  it  to  stand  in  a  cool  place  for  crystallizati-on. 
Examine  the  crystals  under  the  microscope  and  compare  them  with 
those  reproduced  in  Fig.  89,  below. 

4.  Murexide  Test. — To  a  small  amount  of  pure  uric  acid  in  a 
small  evaporating  dish  add  2-3  drops  of  concentrated  nitric  acid. 
Evaporate  to  dryness  carefully  on  a  water-bath  or  over  a  \-ery  low 
flame.  A  red  or  yellow  residue  remains  which  turns  purplish-red 
after  cooling  the  dish  and  adding  a  drop  of  very  dilute  ammonium 
hydroxide.     The  color  is  due  to  the  formation  of  murexide.     If  potas- 


FiG.  8q. — Pure  Uric  Acid. 

slum  hydroxide  is  used  instead  of  ammonium  hydroxide  a  purplish- 
violet  color  due  to  the  production  of  the  potassium  salt  is  obtained. 
The  color  disappears  upon  warming;  with  certain  related  bodies 
(purine  bases)  the  color  persists  under  these  conditions. 

5.  Moreigne's  Reaction. — To  equal  volumes  of  Moreigne's 
reagent^  and  the  solution  to  be  tested  add  a  few  drops  of  concentrated 
potassium  hydroxide.     A  blue  color  indicates  the  presence  of  uric  acid. 

6.  Schiff's  Reaction. — Dissolve  a  small  amount  of  pure  uric  acid 
in  sodium  carbonate  solution  and  transfer  a  drop  of  the  resulting  mix- 
ture to  a  strip  of  filter  paper  saturated  with  argentic  nitrate  solution. 
A  yellowish-brown  or  black  coloration  due  to  the  formation  of  reduced 
silver  is  produced. 

7.  Ganassini's  Test.^ — Dissolve  a  small  amount  of  uric  acid  in 

'  Moreigne's  reagent  is  made  by  combining  20  grams  of  sodium  tungstate,  ro  grams 
of  phosphoric  acid  (sp.  gr.  1.13)  and  100  c.c.  of  water.  Boil  this  mi.xture  for  twenty 
minutes,  add  water  to  make  the  volume  of  the  solution  equivalent  to  the  original  volume, 
and  acidify  with  hydrochloric  acid. 

'Ganassini;  Boll,  soc,  1908,  No.  i. 


270  PHYSIOLOGICAL    CHEMISTRY. 

sodivim  carbonate.  Precipitate  the  dissolved  uric  acid  by  means  of 
zinc  chloride,  filter  off  the  precipitate,  and  permit  it  to  stand  in  contact 
with  the  air.  A  sky-blue  color  will  develop,  a  color  change  which  may 
be  hastened  by  sunlight.  A  similar  reaction  may  be  obtained  by  treat- 
ing the  original  precipitate  with  K,S,0^. 

8.  Influence  upon  Fehling's  Solution. — Dilute  i  c.c.  of  Feh- 
ling's  solution  with  4  c.c.  of  water  and  heat  to  boiling.  Now  add 
slowly,  a  few  drops  at  a  time,  1-2  c.c.  of  a  concentrated  solution  of  uric 
acid  in  potassium  hydroxide,  heating  after  each  addition.  From  this 
experiment  what  do  you  conclude  regarding  .the  possibility  of  arriving 
at  an  erroneous  decision  when  testing  for  sugar  in  the  urine  l)v  means 
of  Fehling's  test  ? 

9.  Reduction  of  Nylander's  Reagent. — To  5  c.c.  of  a  solution  of 
uric  acid  in  potassium  hydroxide  add  about  one-half  a  cubic  centimeter 
of  Xylander's  reagent  and  heat  to  boiling  for  a  few  moments.  Do  you 
obtain  the  typical  black  end-reaction  signifying  the  reduction  of  the 
bismuth  ? 

NH  —  CO 

I 

CREATININE,    C  =  NH 

N.(CH3).CH,. 

Creatinine  is  the  anhydride  of  creatine  and  is  a  constituent  of  normal 
human  urine.  The  theory  that  creatinine  is  deri\ed  from  the  creatine 
of  ingested  muscular  tissue  as  well  as  from  the  creatine  of  the  muscular 
tissue  of  the  organism  has  recently  been  proven  to  be  incorrect  by  Folin, 
Klercker,  and  Wolf  and  Shaffer.  ShalTer  believes  that  creatinine  is 
the  result  of  some  special  ])rocess  of  normal  metalxjlism  which  takes 
place  to  a  large  extent,  if  not  entirely,  in  the  muscles  and  further  that 
the  amount  (A  such  creatinine  elimination,  expressed  in  milligrams  per 
kilogram  body  weight,  is  an  index  of  this  special  process.^  He  further 
states  that  the  muscular  efficiency  of  the  individual  depends  upon  the 
intensity  of  this  process.  Under  normal  conditions  about  i  gram  of 
creatinine  is  excreted  by  an  adult  man  in  24  hours, ^  the  exact  amount 
depending  in  great  ])art  upon  the  nature  of  the  food  and  decreasing 
markedly  in  starvation.  Very  little  that  is  important  is  known  regard- 
ing the  excretion  of  creatinine  under  ])athological  conditions.  The 
creatinine  content  of  the  urine  is  said  to  be  increased  in  typhoid  fever, 

'  He  proposes  to  designate  as  the  "creatinine  <  oclTu  iciit  "  llu-  ex< n-tion  of  creatiuine- 
nitragen  (mgs.)  per  kiloi^ram  of  body  weight. 

-According  tf)  S.hafTer  the  amount  e.x(  retcd  by  strictly  normal  individuals  is  helween 
7  and  II  milligrams  of  <  reatinine-nitrogen  jkt  kilogram  of  body  weight. 


URINE. 


271 


typhus,  tetanus,  and  pneumonia,  and  to  be  decreased  in  ansemia,  chlo- 
rosis, paralysis,  muscular  atrophy,  advanced  degeneration  of  the  kid- 
neys, and  in  leucaemia  (myelogenous,  lymphatic  and  pseudo).  An 
increase  of  creatinine  was  also  noted  in  diabetes,  an  increase  probably 
due  to  the  creatinine  content  of  the  meat  eaten.  The  greater  part  of 
the  data,  however,  relating  to  the  variation  of  the  creatinine  excretion 
under  pathological  conditions  are  not  of  much  value  since  in  nearly 
every  instance  the  diet  was  not  sufficiently  controlled  to  permit  the 
collection  of  reliable  data.  And  further,  until  the  advent  of  the  Folin 
method  (see  p.  385),  there  was  no  accurate  method  for  the  c|uantitative 


Fig.  00. — Creatixink. 


determination  of  creatinine.  Shaffer  has  very  recently  called  attention 
to  the  fact  that  a  low  excretion  of  creatinine  is  found  in  the  urine  of  a 
remarkably  large  number  of  pathological  subjects,  representing  a  vari- 
ety of  conditions,  and  that  it  is  therefore  evident  that  the  excretion  of 
an  abnormally  small  amount  of  this  substance  is  by  no  means  peculiar 
to  any  one  disease. 

Creatinine  crystallizes  in  colorless,  glistening  monoclinic  prisms 
(Fig.  90,  above)  which  are  soluble  in  about  12  parts  of  cold  w^ater; 
they  are  more  soluble  in  warm  water  and  in  warm  alcohol.  One  of  the 
most  important  and  interesting  of  the  compounds  of  creatinine  is  crea- 
tinine-zinc  ciihn'de,  {CJij'N^O)^ZnC\^,  which  is  formed  from  an  alco- 
holic solution  of  creatinine  upon  treatment  with  zinc  chloride  in  acid 
solution.  Creatinine  has  the  power  of  reducing  cupric  hydroxide  in 
alkaline  solution  and  in  this  way  may  interfere  with  the  determination 
of  sugar  in  the  urine.     In  the  reduction  by  creatinine  the  blue  liquid  is 


272  PHYSIOLOGICAL    CHEMISTRY. 

first  changed  to  a  yellow  and  the  formation  of  a  Ijrownish-rcd  precipi- 
tate of  cuprous  oxide  is  brought  about  only  after  continuous  boiling 
with  an  excess  of  the  copper  salt.  Creatinine  does  not  reduce  alkaline 
bismuth  solutions  and  therefore  does  not  interfere  with  Xylander's 
and  Boettger's  tests. 

It  has  recently  been  shown  by  Folin  that  the  absolute  quantity  of 
creatinine  eliminated  in  the  urine  on  a  meat-free  diet  is  a  constant 
quantity  dififerent  for  different  individuals,  but  wholly  independent 
of  quantitative  changes  in  the  total  amount  of  nitrogen  eliminated. 
Shaffer  has  very  recently  confirmed  these  findings  and  has  shown  that 
the  output  of  creatinine  under  these  conditions  is  constant  from  hour 
to  hour  as  well  as  from  day  to  day. 

Experiments  ox  Creatinine. 

I.  Separation  from  the  Urine. — Place  250  c.c.  of  urine  in  a 
casserole  or  beaker,  render  it  alkaline  with  milk  of  lime  and  then  add 
CaClj  solution  until  the  phosphates  are  completely  precipitated. 
Filter  off  the  precipitate,  render  the  filtrate  slightly  acid  with  acetic 
acid,  and  evaporate  it  to  a  syrup.     While  still  warm  this  syrup    is 


Fig.  (>i. — Ckkatim.nk-Zinc  Cmiokidk.     (Sn/kinc.'ski.) 

treated  with  about  50  c.c.  of  95-97  per  cent  alcohol  and  the  mixture 
allowed  to  stand  8-12  hours  in  a  cool  place.  The  precij)itate  is  now 
filtered  off  and  the  filtrate  treated  with  a  little  sodium  acetate  and 
about  one-half  c.c.  of  acid-free  zinc  chloride  solulion  having  a  specific 
gravity  of  1.2.  This  mixture  is  stirred  thoroughly  and  allowed  to 
stand  in  a  cold  place  for  48  72  hours.  Creatinine  zinc  chloride  (Fig. 
91,  abo\ej   will  crystallize  out   under  these  conditions.      Collect    the 


URINE.  273 

crystals  on  a  filter  paper  and  wash  them  with  alcohol  to  remo\e  chlo- 
rides. Now  treat  the  zinc  chloride  compound  with  a  little  warm 
water,  boil  with  lead  oxide  and  filter.  The  filtrate  may  now  be  decol- 
orized by  animal  charcoal,  evaporated  to  dryness,  and  the  residue 
extracted  with  strong  alcohol.  (Creatine  remains  undissolved  under 
these  conditions.)  The  alcoholic  extract  of  creatinine  is  now  e^■apo- 
rated  to  incipient  crystallization  and  left  in  a  cool  place  until  crystal- 
lization is  complete.  If  desired  the  crystals  may  be  purified  by  re- 
crystallization  from  water. 

2.  Weyl's  Test. — Take  5  c.c.  of  urine  in  a  test-tube,  add  a  few 
drops  of  sodium  nitro-prusside  and  render  the  solution  alkaline  with 
potassium  hydroxide  solution.  A  ruby-red  color  results  which  soon 
turns  yellow.     See  Legal 's  test  for  acetone,  page  ;^27i. 

3.  Salkowski's  Test. — -To  the  yellow  solution  obtained  in  Weyl's 
test  above  add  an  excess  of  acetic  acid  and  apply  heat.  A  green  color 
results  and  is  in  turn  displaced  by  a  blue  color.  A  precipitate  of 
Prussian  blue  may  form. 

4.  Jaffe's  Reaction. — Place  5  c.c.  of  urine  in  a  test-tube,  add  an 
aqueous  solution  of  picric  acid  and  render  the  mixture  alkaline  with 
potassium  hydroxide  solution.  A  red  color  is  produced  which  turns 
yellow  if  the  solution  be  acidified.  Dextrose  gives  a  similar  red  color 
but  only  upon  the  application  of  heat.  This  color  reaction  observed 
when  creatinine  in  alkaline  solution  is  treated  with  picric  acid  is  the 
basic  principle  of  Folin's  colorimetric  method  for  the  quantitative 
determination  of  creatinine  (see  page  385). 

ETHEREAL  SULPHURIC  ACIDS. 

The  most  important  of  the  ethereal  sulphuric  acids  found  in  the 
urine  are  phenol- sulphuric  acid,  p-cresol-sulphuric  acid,  indoxyl- sulphuric 
acid,  and  skatoxy I- sulphuric  acid.  Pyrocatechin-sulphuric  acid  also 
occurs  in  traces  in  human  urine.  The  total  output  of  ethereal  sul- 
phuric acid  varies  from  0.09  to  0.62  gram  for  24  houts.  In  health  the 
ratio  of  ethereal  sulphuric  acid  to  inorganic  sulphuric  acid  is  about 
I  :  10.  These  ethereal  sulphuric  acids  originate  in  part  from  the 
phenol,  cresol,  indole  and  skatole  formed  in  the  putrefaction  of  protein 
material  in  the  intestine.  The  phenol  passes  to  the  liver  where  it  is 
conjugated  to  form  phenol  potassium  sulphate  and  appears  in  this 
form  in  the  urine  whereas  the  indole  and  skatole  undergo  a  preliminary 
oxidation  to  form  indoxyl  and  skaioxyl  respectively  before  their 
elimination. 
18 


2  74  PHYSIOLOGICAL    CHEMISTRY. 

It  has  generally  been  considered  that  each  of  the  ethereal  sul- 
phuric acids  was  formed  principally  in  the  putrefaction  of  protein 
material  in  the  intestine  and  that  therefore  a  determination  of  the 
total  ethereal  sulphuric  acid  content  of  the  urine  was  an  index  of  the 
extent  to  which  these  putrefactive  processes  were  proceeding  within 
the  organism.  Recently,  however,  Folin  has  conducted  a  series  of 
experiments  which  seem  to  show  that  the  ethereal  sulphuric  acid  con- 
tent of  the  urine  does  not  afford  an  index  of  the  extent  of  intestinal 
putrefaction,  since  these  bodies  arise  only  in  part  from  putrefactive 
processes.  He  claims  that  the  ethereal  sulphuric  acid  excretion 
represents  a  form  of  sulphur  metabolism  which  is  more  in  evidence 
upon  a  diet  containing  a  ^■ery  small  amount  of  protein  or  upon  a  diet 
containing  absolutely  no  protein.  The  ethereal  sulphuric  acid  content 
of  the  urine  diminishes  as  the  total  sulphur  content  diminishes  but 
the  percentage  decrease  is  much  less.  Therefore  when  considered 
from  the  standpoint  of  the  total  sulphuric  acid  content  the  ethereal 
sulphuric  acid  content  is  not  diminished  but  is  increased,  although  the 
total  sulphuric  acid  content  is  diminished.  Folin 's  experiments  also 
seem  to  show  that  the  indoxyl  sulphuric  acid  (indoxyl  potassium 
sulphate  or  indican)  content  of  the  urine  does  not  originate  to  any 
degree  from  the  metabolism  of  protein  material  but  that  it  arises  in 
great  part  from  intestinal  putrefaction  and  that  the  excretion  of  indoxyl 
sulphuric  acid  may  alone  be  taken  as  a  rough  index  of  the  extent  of 
putrefactixe  processes  within  the  intestine.     Indoxyl  sulphuric  acid, 

CH 


HC        C-C(O.SO.,H), 

I      !I     I! 

HC       C      CH 
CH  NH 

therefore,    which   occurs  in   the   urine  as  indoxyl   potassium   suljjhale 
or    indican, 

CH 

HC        C- C(().S(),,Rj, 

I         II        II 
HC       C      CH 

\/\/ 
CH  Nil 

is  clinicalU-  the  most  imporlaiit  of  the  cllicrcal  siilpluiric  acids. 


URINE.  275 

Tests  for  Indicax. 

I.  Jaffe's  Test. — Nearly  fill  a  test-tube  with  a  mixture  composed 
of  equal  volumes  of  concentrated  HCl  and  the  urine  under  exam- 
ination. Add  2-3  c.c.  of  chloroform  and  a  few  drops  of  a  calcium 
hypochlorite  solution,  place  the  thumb  over  the  end  of  the  test-tube 
and  shake  the  tube  and  contents  thoroughly.  The  chloroform  is 
colored  more  or  less,  according  to  the  amount  of  indican  present. 
Ordinarily  a  blue  color  due  to  the  formation  of  indigo-blue  is  pro- 
duced; less  frequently  a  red  color  due  to  indigo-red  may  be  noted. 

This  is  the  reaction  (see  also  pages  158  and  159): 

CH 


HC       C-COH 

'■     \         II       li  +20  = 

HC       C      CH 


CH  NH 

Indoxyl.  CsHtNO. 

CH  CH 

/\  /"X 

HC       C-CO      O.C-C       CH 

I  II         i  I         II  I        _L  oH  o 

HC       C      C C      C       CH 

/  \ 


CH    NH  NH   CH 

Indigo-blue.  C15H10N2O:. 

2.  Obermayer's  Test. — Nearly  fill  a  test-tube  with  a  mixture 
composed  of  equal  volumes  of  Obermayer's  reagent^  and  the  urine 
under  examination.  Add  2-3  c.c.  of  chloroform,  place  the  thumb 
over  the  end  of  the  test-tube  and  shake  thoroughly.  How  does  this 
compare  with  Jaffe's  test? 

3.  Giirber's  Reaction. — To  one  volume  of  the  urine  under 
examination  and  two  volumes  of  concentrated  hydrochloric  acid  in  a 
test-tube  add  2-3  drops  of  a  i  per  cent  solution  of  osmic  acid  and 
2-3  c.c.  of  chloroform  and  shake  the  tube  and  contents  thoroughly. 
Compare  the  color  with  those  obtained  in  Jaffe's  and  Obermayer's  tests. 

An  excess  of  osmic  acid  does  not  affect  the  reaction.  Occasionally 
better  results  are  obtained  if  the  solution  of  osmic  acid  is  added  directly 
to  the  urine  before  the  addition  of  the  hydrochloric  acid.  If  the  urine 
under  examination  be  strongly  colored  or  of  high  specific  gravity  it 
should  first  be  treated  with  basic  lead  acetate  (one-eighth  volume). 

1  Obermayer's  reagent  is  prepared  by  adding  2-4  grams  of  ferric  chloride  to  a  liter  of 
concentrated  HCl  (sp.  gr.  i.iq). 


2/6 


PHYSIOLOGICAL    CHEMISTRY. 


The  precipitate  is  then  removed  by  fihration  and  the  resuhing  tihrate 
used  in  making  the  test  for  indican. 

4.  Rossi's  Reaction. — To  equal  volumes  of  concentrated  hydro- 
chloric acid  and  the  urine  under  examination,  in  a  test-tube,  add  i 
drop  of  a  10  per  cent  solution  of  ammonium  persulphate  and  2-3  c.c. 
of  chloroform.  Agitate  the  mixture  vigorously  and  note  the  color  of 
the  chloroform.  Compare  this  result  with  those  obtained  in  the  other 
indican  tests. 

;.  Lavelle's  Reaction. — To  10  c.c.  of  urine  in  a  test-tube  add 
2-3  c.c.  of  Obermayer's  reagent^  and  a  similar  volume  of  concen- 
trated sulphuric  acid.  (During  the  addition  of  the  acid  the  tube 
should  be  held  under  running  water  in  order  that  the  temperature  of 
the  mixture  may  not  rise  too  high.)  Add  2-3  c.c.  of  chloroform, 
shake  the  tube  vigorously,  and  observe  the  depth  of  color  assumed  by 
the    chloroform. 

The  sponsor  for  this  reaction  claims  it  to  be  the  most  satisfactory 
of  the  indican  tests. 

CO.NH.CH,.COOH. 

HIPPURIC  ACID, 

This  acid  occurs  normally  in  the  urine  of  both  the  carnivora  and 
herbi\"ora  but  is  more  abundant  in  the  urine  of  the  latter.     It  is  formed 


Fio.  t;2.     IIiiM'URic  Acid. 

by  a  synthesis  of  benzoic  acid  and  giycocoll  wliic  h  takes  jjlace  in  the 
kidneys.     The  average  excretion  of  an  aduli  man  for  24  hours  under 

'  Obermayer's  reagent  is  prepared  hy  ad.Mn;'  2-4  grams  of  fc-rric  cliloride  lo  ;i.  liter  oi 
concentrated  PICl  (sp.  gr.  i .  ly). 


URINE.  277 

normal  conditions  is  about  0.7  gram.  Hippuric  acid  crystallizes  in 
needles  or  rhombic  prisms  (see  Fig.  92,  p.  276)  the  particular  form 
depending  upon  the  rapidity  of  crystallization.  Pure  hippuric  acid 
melts  at  187°  C.  The  most  satisfactory  method  for  the  isolation  of 
hippuric  acid  from  the  urine  in  crystalline  form  is  that  proposed  by 
Roaf  (see  below).  It  is  easily  soluble  in  alcohol  or  hot  water,  and 
only  slightly  soluble  in  ether.  The  output  of  hippuric  acid  is  increased 
in  diabetes  owing  probably  to  the  ingestion  of  much  protein  and  fruit. 
It  is  decreased  in  fevers  and  in  certain  kidney  disorders  where  the 
synthetic  activity  of  the  renal  cells  is  diminished.  Hippuric  acid  may 
be  determined  quantitatively  by  means  of  Dakin's  methods  (see 
p.  376). 

Experiments  on  Hippuric  Acid. 

I.  Separation  from  the  Urine,  (a)  First  Method. — Render  500- 
1000  c.c.  of  urine  of  the  horse  or  cow^  alkaline  with  milk  of  lime, 
boil  for  a  few  moments  and  filter  while  hot.  Concentrate  the  filtrate, 
over  a  burner,  to  a  small  volume.  Cool  the  solution,  acidify  it  strongly 
with  concentrated  hydrochloric  acid  and  stand  it  in  a  cool  place  for 
24  hours.  Filter  off  the  crystals  of  hippuric  acid  which  have  formed 
and  wash  them  wnth  a  little  cold  water.  Remove  the  crystals  from 
the  paper,  dissolve  them  in  a  very  small  amount  of  hot  water  and  per- 
colate the  hot  solution  through  thoroughly  washed  animal  charcoal, 
being  careful  to  wash  out  the  last  portion  of  the  hippuric  acid  solution 
with  hot  water.  Filter,  concentrate  the  filtrate  to  a  small  volume  and 
stand  it  aside  for  crystallization.  Examine  the  crystals  under  the 
microscope  and  compare  them  with  those  in  Fig.  92,  page  276.  This 
method  is  not  as  satisfactory  as  Roaf 's  method  (see  below). 

{h)  Roafs  Method.^ — ^Place  500  c.c.  of  urine  of  the  horse  or  cow^ 
in  a  casserole  or  precipitating  jar  and  add  an  equal  volume  of  a  satu- 
rated solution  of  ammonium  sulphate^  and  7.5  c.c.  of  concentrated 
sulphuric  acid.  Permit  the  mixture  to  stand  for  twenty-four  hours  and 
remove  the  crystals  of  hippuric  acid  by  filtration.  Purify  the  crystals 
by  recrystallization  according  to  the  directions  given  above  under  First 
Method.  Examine  the  crystals  under  the  microscope  and  compare 
them  with  those  given  in  Fig.  92  p.  276. 

^  If  urine  of  the  horse  or  cow  is  not  available  human  urine  may  serve  the  purpose  fully 
as  well  provided  means  are  taken  to  increase  its  content  of  hippuric  acid.  This  may  be 
conveniently  accomplished  by  ingesting  2  grams  of  ammonium  benzoate  at  night.  The 
fraction  of  urine  passed  in  the  morning  will  be  found  to  have  a  high  content  of  hippuric 
acid.     The  ammonium  benzoate  is  in  no  wa)*  harmful. 

-  125  grams  of  solid  ammonium  sulphate  may  be  substituted. 


2-jS  PHYSIOLOGICAL    CHEMISTRY. 

If  sufficient  urine  is  not  available  to  permit  the  use  of  500  c.c. 
a  smaller  volume  may  be  used  inasmuch  as  it  is  possible,  by  the  above 
technique,  to  isolate  hippuric  acid  in  crystalline  form  from  as  small  a 
volume  as  25-50  c.c.  of  herbivorous  urine.  The  greater  the  amount  of 
ammonium  sulphate  added  the  more  rapid  the  crystallization  until 
at  the  saturation  point  the  crystals  of  hippuric  acid  sometimes  form  in 
about  ten  mimiles. 

2.  Melting-point. — Determine  the  melting-point  of  the  hippuric 
acid  prepared  in  the  above  experiment  (see  p.  264). 

3.  Solubility. — Test  the  solubility  of  hippuric  acid  in  the  ordi- 
narv  solvents  (page  22)  and  in  alcohol,  and  ether. 

4.  Dehn.'s  Reaction. — Introduce  about  5  c.c.  of  the  urine  or  the 
solution  under  examination  into  a  test-tube  and  add  sufficient  hypo- 
bromite  solution^  to  impart  to  the  mixture  a  permanent  yellow  color. 
In  the  case  of  urine  enough  hypobromite  should  be  added  to  decom- 
pose the  urea.  Heat  the  mixture  to  boiling  and  note  the  formation 
of  an  orange  or  brown-red  precipitate  if  hippuric  acid  is  present.  If 
the  solution  under  examination  contains  only  a  trace  of  hippuric  acid 
the  solution  will  appear  smoky  and  faintly  red  in  color,  whereas  if  a 
larger  amount  of  the  acid  be  present  the  solution  will  become  opaciue 
and  of  an  orange  or  brown-red  color.  In  either  case  after  standing 
for  some  time  the  solution  should  clear  up  and  a  light,  finely  divided 
precipitate  should  be  deposited.  This  precipitate  consists  of  earthy 
phosphates  mixed  with  an  amorphous  orange  or  brown-red  substance 
of  unknown  composition. 

5.  Formation  of  Nitro-Benzene. — To  a  little  hippuric  acid  in 
a  small  porcelain  dish  add  12  c.c.  of  concentrated  HNO3  and  evapo- 
rate to  dryness  on  a  water-bath.  Transfer  the  residue  to  a  dry  test- 
tube,  apply  heat,  anrl  note  the  odor  of  the  artificial  oil  of  l)itler  almonds 
(nitro-ben/ene). 

6.  Sublimation.  I'lace  a  few  crystals  of  hi|)puric  acid  in  a  dry 
test-tube  and  appiv  heat.  The  crystals  are  reduced  to  an  oily  thiid 
which  solidifies  in  a  crystalline  mass  upon  cooling.  When  stronger 
heat  is  applied  the  liquid  assumes  a  red  color  and  fmally  yields  a  sub- 
limate of  ben/.oic  acid  and  the  odor  of  hydrocyanic  acid. 

7.  Formation  of  Ferric  Salt.  Render  a  small  amount  of  a  solu- 
tion i){  hijjjniric  acid  neutral  with  dilute  potassium  hydroxide.  Now 
add  I  3  drops  of  neutral  ferric  chloride  solution  and  note  the  forma- 
tion of  the  ferric  salt  of  hi|)|)iiri(    ac  id  as  a  cream  colored  i)recipitate. 

'  .S<fc  note  on  p.  .369. 


URINE.  279 

COOH 

OXALIC  ACID,    I 

COOH. 

Oxalic  acid  is  a  constituent  of  normal  urine,  about  0.02  gram  being 
eliminated  in  24  hours.  It  is  present  in  the  urine  as  calcium  oxalate, 
which  is  kept  in  solution  through  the  medium  of  the  acid  phosphates. 
The  origin  of  the  oxalic  acid  content  of  the  urine  is  not  well  under- 
stood. It  is  ehminated,  at  least  in  part,  unchanged  when  ingested, 
therefore  since  many  of  the  common  articles  of  diet,  e.  g.,  asparagus, 
apples,  cabbage,  grapes,  lettuce,  spinach,  tomatoes,  etc.,  contain 
oxalic  acid  it  seems  probable  that  the  ingested  food  supplies  a  por- 
tion of  the  oxalic  acid  found  in  the  urine.  There  is  also  experimental 
evidence  that  part  of  the  oxalic  acid  of  the  urine  is  formed  within  the 
organism  in  the  course  of  protein  and  fat  metabolism.  It  has  also 
been  suggested  that  oxalic  acid  may  arise  from  an  incomplete  com- 
bustion of  carbohydrates,  especially  under  certain  abnormal  con- 
ditions. Pathologically,  oxalic  acid  is  found  to  be  increased  in  amount 
in  diabetes  mellitus,  in  organic  diseases  of  the  liver,  and  in  various  other 
conditions  which  are  accompanied  by  a  derangement  of  the  oxidation 
mechanism.  An  abnormal  increase  of  oxalic  acid  is  termed  oxaluria. 
A  considerable  increase  in  the  content  of  oxalic  acid  may  be  noted 
unaccompanied  by  any  other  apparent  symptom.  Calcium  oxalate 
crystallizes  in  at  least  two  distinct  forms,  dumb-hells  and  octahedra 
(Fig.  99,  page  340). 

Experiments. 

Preparation  of  Calcium  Oxalate. — First  Method. — Place  200- 
250  c.c.  of  urine  in  a  beaker,  add  5  c.c.  of  a  saturated  solution  of  cal- 
cium chloride,  make  the  urine  slightly  acid  with  acetic  acid,  and  stand 
the  beaker  aside  in  a  cool  place  for  24  hours.  Examine  the  sediment 
under  the  microscope  and  compare  the  crystalline  forms  with  those 
shown  in  Fig.  99,  p.  340. 

Second  Method. — Proceed  as  above,  replacing  the  acetic  acid  by 
an  excess  of  ammonium  hydroxide  and  filtering  off  the  precipitate  of 
phosphates. 

NEUTRAL  SULPHUR  COMPOUNDS. 

Under  this  head  may  be  classed  such  bodies  as  cystine  (see  p.  70), 
chondroitin-sulphuric  acid,  oxyproteic  acid,  alloxyproteic  acid, 
uroferric   acid,   thiocyanates,   and   taurine  derivatives.     The  sulphur 


2  So 


PHYSIOLOGICAL    CHEMISTRY. 


content  of  the  bodies  just  enumerated  is  generally  termed  loosely 
combined  or  neutral  sulphur  in  order  that  it  may  not  be  confused  with 
the  acid  sulphur  which  occurs  in  the  inorganic  sulphuric  acid  and 
ethereal  sulphuric  acid  forms.  Ordinarily  the  neutral  sulphur  content 
of  normal  human  urine  is  14-20  per  cent  of  the  total  sulphur  content. 

NH.CH.HN 


ALLANTOIN,    QC 


CO. 


NH.CONH3 

Allantoin  has  been  found  in  the  urine  of  suckling  calves  as  well 
as  in  that  of  the  dog  and  cat.  It  has  also  been  detected  in  the  urine 
of  infants  within  the  first  eight  days  after  birth,  as  well  as  in  the  urine 
of  adults.  It  is  more  abundant  in  the  urine  of  women  during  preg- 
nancy.    Underbill  also  reports  the  presence  of  allantoTn  in  the  urine 


KiG.  g^.—  Al.l.A.NTOI.N,    I'KOM  CAT'S  UrINK. 

a  and  b,  Forms  in  which  it  crystallized  fioni  tlic  Lirine;  c,  iccrvNlalli/Ad  all.iiili)iii. 
(Drawn  from  micro  photographs  furni-hi-<l  l.>  I'n.l'.  Lafayette  B.  Meml.l  <>t  ^■al(■ 
University.) 

of  fasting  dogs,  an  observation  which  makes  it  i)robable  thai  allantoin 
is  a  constant  constituent  of  the  urine  of  such  animals.  Allantoin  is 
formed  by  the  oxidation  of  uric  acid  and  ih  output  is  increased  by 
thymus  or  pancreas  feeding.  When  j)urc  it  crystallizes  in  prisms 
(PMg.  9.5.  above)  and  when  impure  in  graiuiles  and  knobs.  Patho- 
logically, it  has  been  f(nmfl  increased  in  diabetes  insipidus  and  in 
hysteria  with  convulsions  (i'ouchet).  Mendel  and  Dakin*  have 
recently  shown   that   allantoin    is   optically   inactive    notwithstanding 

'  .Menflel  an<l  I>al<in:     Jour,  liiol.  C'hcm.,  \ll,  \>.  J.S.i,  i(;io. 


URINE.  281 

the  fact   that  it   contains   an   asymmetric   carbon  atom.     This    phe- 
nomenon they  believe  to  be  due  to  tautomeric  change. 

Experiments. 

1.  Separation  from  the  Urine/ — Meissner's  Method. — Precipi- 
tate the  urine  with  baryta  water.  Neutralize  the  j&ltrate  carefully 
with  dilute  sulphuric  acid,  filter  immediately,  and  evaporate  the  fil- 
trate to  incipient  crystallization.  Completely  precipitate  this  warm 
fluid  with  95  per  cent  alcohol  (reserve  the  precipitate).  Decant  or 
filter  and  precipitate  the  solution  by  ether.  Combine  the  ether  and 
alcohol  precipitates  and  extract  with  cold  water  or  hot  alcohol;  allan- 
toin  remains  undissolved.  Bring  the  allantoin  into  solution  in  hot 
water  and  recrystallize. 

Allantoin  may  be  determined  quantitatively  by  the  Paduschka- 
Underhill-Kleiner  method  (see  p.  401)  or  by  Loewi's  method.^ 

2.  Preparation  from  Uric  Acid. — Dissolve  4  grams  of  uric  acid 
in  100  c.c.  of  water  rendered  alkaline  with  potassium  hydroxide. 
Cool  and  carefully  add  3  grams  of  potassium  permanganate.  Filter, 
immediately  acidulate  the  filtrate  with  acetic  acid  and  allow  it  to  stand 
in  a  cool  place  over  night.  Filter  off  the  crystals  and  wash  them  with 
water.  Save  the  wash  water  and  filtrate,  unite  them  and  after  concen- 
trating to  a  small  volume  stand  away  for  crystallization.  Now  com- 
bine all  the  crystals  and  recrystallize  them  from  hot  water.  Use  these 
crystals  in  the  experiments  which  follow. 

3.  Microscopical  Examination. — Examine  the  crystals  made 
in  the  last  experiment  and  compare  them  with  those  shown  in  Fig. 
93,  page  280. 

4.  Solubility. — Test  the  solubility  of  allantoin  in  the  ordinary 
solvents  (page  22.) 

5.  Reaction. — Dissolve  a  crystal  in  water  and  test  the  reaction 
to  litmus. 

6.  Furfurol  Test. — Place  a  few  crystals  of  allantoin  on  a  test- 
tablet  or  in  a  porcelain  dish  and  add  1-2  drops  of  a  concentrated 
aqueous  solution  of  furfurol  and  1-2  drops  of  concentrated  hydro- 
chloric acid.  Observe  the  formation  of  a  yellow  color  which  turns 
to  a  light  purple  if  allowed  to  stand.  This  test  is  given  by  urea  but 
not  by  uric  acid. 

7.  Murexide  Test. — Try  this  test  according  to  the  directions 
given  on  page  269.     Note  that  allantoin  fails  to  respond. 

'  The  urine  of  the  dog  after  thymus,  pancreas,  or  uric  acid  feeding  may  be  employed. 
-  Archiv  fiir  Experimentelle  Pathologie  und  Pharmakologie,  1900,  XLIV,  p.  20. 


252  PHYSIOLOGICAL    CHEMISTRY. 

8.  Reduction  of  Fehling's  Solution. — Alake  this  test  in  the 
usual  way  (see  p.  27)  except  that  the  boiling  must  be  prolonged  and 
excessive.  Ultimately  the  allantoin  will  reduce  the  solution.  Com- 
pare with  the  result  on  uric  acid,  page  270. 

AROMATIC  OXYACIDS. 

Two  of  the  most  important  of  the  oxyacids  arc  paraoxyphenyl- 
acelic  acid, 

CH^.COOH, 

/\ 


OH 

and  paraoxy phenyl- propionic  acid. 


CH,.CH.,.COOH. 

/\ 


OH 


They  are  products  of  tlie  putrefaction  of  jjrotein  niakTial  and  tyro- 
sine is  an  intermediate  stage  in  their  formation,  iioth  these  acids 
for  the  most  part  pass  unchanged  into  the  urine  where  they  occur 
normally  in  very  small  amount.  The  content  may  be  increased  in 
the  same  manner  as  the  phenol  content,  in  particular  by  acute  phos- 
phorous poisoning.  A  fraction  of  the  total  aromatic  oxyacid  content 
of  the  urine  is  in  combination  with  sulphuric  acid,  but  the  greater 
part  is  present  in  the  form  of  salts  of  sodium  and  potassium. 
Ilomogentisic  Acid  or  di-oxyphenyl-acetic  acid, 

OH 

CH,.COOH, 


OH 

is  another  imijorlant  oxyacid  sometimes  jjresent  in  the  urine.  Under  the 
name  glyco.suric  acid  it  was  first  isolated  from  the  urine  by  f'rof.  John 
Marshall  of  the  University  of  Pennsylvania;  sul)S(<|ucntly  Haumann 
isolated  it  and  determined  its  chemical  constiliilioii.  It  occurs  in 
cases  of  alcaplomiria.  A  urine  containing  this  oxyacid  turns  greenish- 
brown  from  the  surface  downward  when  treated  with  a  little  sodium 


URINE.  283 

hydroxide   or  ammonia.     If  the  sohition  be  stirred   the   color   \-ery 
soon  becomes  dark  brown  or  even  black.     Homogentisic  acid  reduces 
alkaline  copper  solutions  but  not  alkaline  bismuth  solutions.      Urol- 
eucic  acid  is  similar  in  its  reactions  to  homogentisic  acid. 
Oxymandelic  Acid  or  paraoxyphenyl-glycolic  acid, 

OH 


CH(OH).COOH, 

has  been  detected  in  the  urine  in  cases  of  yellow  atrophy  of  the  liver. 
Kynurenic  Acid  or  /--oxy-.j-quinoline  carbonic  acid, 

CH     COH 

/\/\ 
HC        C       CCOOH, 

HC        C       CH 

CH    N 

is  present  in  the  urine  of  the  dog  and  has  recently  been  detected  by 
Swain  in  the  urine  of  the  coyote.  To  isolate  it  from  the  urine  proceed 
as  follows:  Acidify  the  urine  with  hydrochloric  acid  in  the  propor- 
tion 1:25.  From  this  acid  fluid  both  the  uric  acid  and  the  kynurenic 
acid  separate  in  the  course  of  24-48  hours.  Filter  off  the  combined 
crystalline  deposit  of  the  two  acids,  dissolve  the  kynurenic  acid  in  dilute 
ammonia  (uric  acid  is  insoluble),  and  reprecipitate  i't  with  hydrochloric 
acid. 

Kynurenic  acid  may  be   C[uantitatively  determined  by   Capaldi's 
method.^ 

COOH. 

BENZOIC  ACID, 


Benzoic  acid  has  been  detected  in  the  urine  of  the  rabbit  and  dog. 
It  is  also  said  to  occur  in  human  urine  accompanying  renal  disor- 
ders. The  benzoic  acid  probably  originates  from  a  fermentative 
decomposition  of  the  hippuric  acid  of  the  urine. 

'  Zeitschrift  fiir  physiologische  Chcmie,  1897,  XXIII,  p.  92. 


284 


physiological  chemistry. 
Experiments. 


1.  Solubility. — Test  the  solubility  of  benzoic  acid  in  water,  alco- 
hol, and  ether. 

2.  Crystalline  Form. — Recrystallize  some  benzoic  acid  from  hot 
water,  examine  the  crystals  under  the  microscope,  and  compare  them 
with  those  reproduced  in  Fig.  94,  below. 

3.  Sublimation. — Place  a  little  benzoic  acid  in  a  test-tube  and 
heat  over  a  tlame.  Note  the  odor  which  is  evolved  and  observe  that 
the  acid  sublimes  in  the  form  of  needles. 


Fig.  94.     Benzoic  .'\cid. 

4.  Dissolve  a  little  sodium  benzoate  in  water  and  add  a  solution 
of  neutral  ferric  chloride.  Note  the  production  of  a  brownish-yellow 
precipitate  (salicylic  acid  gives  a  reddish-violet  color  under  the  same 
conditions).  Add  ammonium  hydrcjxide  to  some  of  the  precipitate. 
It  dissolves  and  ferric  hydroxide  is  formed.  Add  a  little  hydrochloric 
acid  to  another  portion  of  the  original  precipitate  and  stand  the  vessel 
away  over  night.     What  do  you  observe  ? 


NUCLEOPROTEIN. 

The  nubecula  of  normal  urine  has  been  shown  by  one  investigator 
to  consist  of  a  mucoid  containing  12.7  per  cent  of  nitrogen  and  2.3 
per  cent  of  sulphur.  This  body  evidently  originates  in  the  uri- 
nary passages.  It  is  probably  slightly  soluble  in  the  urine.  Some 
investigators  believe  that  the  body  forming  the  nubecula  of  normal 
urine  is  nucleoprotein  and  not  a  mucin  or  mueoifl  as  stated  above. 


URINE.  285 

A  discussion  of  nucleoprotein  and  related  bodies    occurring    in  the 
urine  under  pathological  conditions  will  be  found  on  page  315. 

NH-CO 

OXALURIC  ACID,    CO 

NH3    COOH. 

Oxaluric  acid  is  not  a  constant  constituent  of  normal  human  urine, 
and  when  found  occurs  only  in  traces  as  the  ammonium  salt.  Upon 
boiling  oxaluric  acid  it  splits  into  oxalic  acid  and  urea. 

ENZYMES. 

Various  types  of  enzymes  produced  within  the  organism  are  ex- 
creted in  both  the  feces  and  the  urine.  In  this  connection  it  is  interest- 
ing to  note  that  pepsin,  gastric  rennin,  and  an  amylase  ha^■e  been  posi- 
tively identified  in  the  urine.  The  occurrence  of  trypsin  in  the  urine, 
at  least  under  normal  conditions,  is  questioned. 

VOLATILE  FATTY  ACIDS. 

Acetic,  butyric,  and  formic  acids  have  been  found  under  normal 
conditions  in  the  urine  of  man  and  of  certain  carnivora  as  well  as 
in  the  urine  of  herbivora.  Normally  they  arise  principally  from 
the  fermentation  of  carbohydrates  and  the  putrefaction  of  proteins. 
The  acids  containing  the  fewest  carbon  atoms  (formic  and  acetic) 
are  found  to  be  present  in  larger  percentage  than  those  which  contain 
a  larger  number  of  such  atoms.  The  volatile  fatty  acids  occur  in 
normal  urine  in  traces,  the  total  output  for  twenty-four  hours,  accord- 
ing to  different  investigators,  varying  from  0.008  gram  to  0.05  gram. 

Pathologically,  the  excretion  of  volatile  fatty  acids  is  increased 
in  diabetes,  fevers,  and  in  certain  hepatic  diseases  in  which  the  paren- 
chyma of  the  liver  is  seriously  affected.  Under  other  pathological 
conditions  the  output  may  be  diminished.  These  variations,  however, 
in  the  excretion  of  the  volatile  fatty  acids  possess  very  little  diagnostic 
value. 

CH3 

PARALACTIC  ACID,    CH{OH) 

COOH. 


286  PHYSIOLOGICAL    CHEMISTRY. 

Paralactic  acid  is  supposed  to  pass  into  the  urine  \Yhen  the  supply 
of  oxygen  in  the  organism  is  diminished  through  any  cause,  e.  g., 
after  acute  yellow  atrophy  of  the  liver,  acute  phosphorus  poisoning, 
or  epileptic  attacks.  This  acid  has  also  been  found  in  the  urine  of 
healthy  persons  following  the  physical  exercise  incident  to  prolonged 
marching.  Paralactic  acid  has  been  detected  in  the  urine  of  birds 
after  the  removal  of  the  liver.  Underhill  reports  the  occurrence  of 
this  acid  in  the  urine  of  a  case  of  pernicious  vomiting  of  pregnancy. 

CH.,CO.NH.CH...COOH. 

PHENACETURIC  ACID,     I 


Phenaceturic  acid  occurs  principally  in  the  urine  of  herbixorous 
animals  but  has  frequently  been  detected  in  human  urine.  It  is  pro- 
duced in  the  organism  through  the  synthesis  of  glycocoll  and  phenyl- 
acetic  acid.  It  may  be  decomposed  into  its  component  parts  by  boiling 
with  dilute  mineral  acids.  The  crystalline  form  of  phenaceturic  acid 
(small  rhombic  plates  with  rounded  angles)  resembles  one  form  of 
uric  acid  crystal. 

PHOSPHORIZED  COMPOUNDS. 

Phosphorus  in  organic  combination  has  been  found  in  the  urine 
in  such  bodies  as  glycerophosphoric  acid,  which  may  arise  from  the 
decomposition  of  lecithin,  and  phosphocarnic  acid.  It  is  claimed 
that  on  the  average  about  2.5  per  cent  of  the  total  phosphorus  elimi- 
nation is  in  organic  coml^ination. 

PIGMENTS. 

There  are  at  least  three  pigments  normally  present  in  human  urine. 
These  pigments  are  urochrome,  urobilin,  and  uroerylhrin. 

A.  UROCHROME. 

This  is  the  {)rinci{)al  ])igmenl  of  normal  urine  and  im])arts  (he 
characteristic  yellow  color  to  that  lluid.  It  is  apparently  closely  related 
to  its  associated  jjigmenl  urobilin  sim c  tlic  Jailer  may  be  readily  con- 
\ertcd  into  urochrome  through  evaporation  iA  its  acjueous-ether  solu- 
tion. Urochrome  may  be  obtained  in  the  form  of  a  brown,  amorphous 
powder  which  is  readily  solul^le  in  water  and  95  per  cent  alcohol.  It 
is  less  soluble  in   absolute  iilcohol,  acetone,  amyl  alcohol,  and  acetic 


URINE.  287 

ether  and  insoluble  in  benzene,  chloroform,  and  ether.     Urochrome  is 
said  to  be  a  nitrogenous  body  (4.2  per  cent  nitrogen),  free  from  iron. 

B.  UROBILIN. 

Urobilin,  which  was  at  one  time  considered  to  be  the  principal 
pigment  of  urine,  in  reality  contributes  little  toward  the  pigmentation 
of  this  fluid.  It  is  claimed  that  no  urobilin  is  present  in  freshly  voided 
normal  urine  but  that  its  precursor,  a  chromogen  called  urobilinogen, 
is  present  and  gives  rise  to  urobilin  upon  decomposition  through  the 
influence  of  light.  It  is  claimed  by  some  investigators  that  there  are 
various  forms  of  urobilin,  e.  g.,  normal,  febrile,  physiological,  and 
pathological.  Urobilin  is  said  to  be  very  similar  to,  if  not  absolutely 
identical  with,  hydrobilirubin  (see  page  169). 

Urobilin  may  be  obtained  as  an  amorphous  powder  which  varies 
in  color  from  brown  to  reddish-brown,  red  and  reddish-yellow,  depend- 
ing upon  the  way  in  which  it  is  prepared.  It  is  easily  soluble  in  ethyl 
alcohol,  amyl  alcohol,  and  chloroform,  and  slightly  soluble  in  ether, 
acetic  ether,  and  in  water.  Its  solutions  show  characteristic  absorption- 
bands  (see  Absorption  Spectra,  Plate  II).  Under  normal  conditions 
urobilin  is  derived  from  the  bile  pigments  in  the  intestine. 

Urobilin  is  increased  in  most  acute  infectious  diseases  such  as 
erysipelas,  malaria,  pneumonia,  and  scarlet  fever.  It  is  also  increased 
in  appendicitis,  carcinoma  of  the  liver,  catarrhal  icterus,  pernicious 
ancemia,  and  in  cases  of  poisoning  by  antifebrin,  antipyrin,  pyridin, 
and  potassium  chlorate.  In  general  it  is  usually  increased  when 
blood  destruction  is  excessive  and  in  many  disturbances  of  the  liver. 
It  is  markedly  decreased  in  phosphorus  poisoning. 

Experiments. 

I.  Spectroscopic  Examination. — Acidify  the  urine  with  hydro- 
chloric acid  and  allow  it  to  remain  exposed  to  the  air  for  a  few  moments. 
By  this  means  if  any  urobilinogen  is  present  it  will  be  transformed 
into  urobilin.  The  urine  may  now  be  examined  by  means  of  the 
spectroscope.  If  urobilin  is  present  in  the  fluid  the  characteristic 
absorption-band  lying  between  h  and  F  will  be  observed  (see  Absorp- 
tion Spectra,  Plate  II).  It  may  be  found  necessary  to  dilute  the  urine 
with  water  before  a  distinct  absorption-band  is  observed.  This  test 
may  be  modified  by  acidifying  10  c.c.  of  urine  with  hydrochloric  acid 
and  shaking  it  gently  with  5  c.c.  of  amyl  alcohol.  The  alcoholic 
extract  Avhen  examined  spectroscopically  will  show  the  characteristic 


2SS  PHYSIOLOGICAL    CHEMISTRY. 

urobilin  absorption-band.      (Note   the   spectroscopic   examination   in 
the  next  experiment.) 

2.  Ammoniacal-zinc  Chloride  Test. — Render  some  of  the  urine 
ammoniacal  by  the  addition  of  ammonium  hydroxide,  and  after 
allowing  it  to  stand  a  short  time  filter  off  the  precipitate  of  phosphates 
and  add  a  few  drops  of  zinc  chloride  solution  to  the  filtrate.  Observe 
the  production  of  a  greenish  fluorescence.  Examine  the  fluid  by 
means  of  the  spectroscope  and  note  the  absorption-band  which  occu- 
pies much  the  same  position  as  the  absorption-band  of  urobilin  in  acid 
solution  (sec  Absorption  Spectra,  Plate  II). 

3.  Gerhardt's  Test. — To  20  c.c.  of  urine  add  3-5  c.c.  of  chloro- 
form and  shake  well.  Separate  the  chloroform  extract  and  add  to 
it  a  few  drops  of  iodine  solution  (I  in  KI).  Render  the  mixture  alka- 
line with  dilute  solution  of  potassium  hydroxide  and  note  the  produc- 
tion of  a  yellow  or  yellowish-brown  color.  The  solution  ordinarily 
exhibits  a  greenish  fluorescence. 

4.  Wirsing's  Test. — To  20  c.c.  of  urine  add  3-5  c.c.  of  chloro- 
form and  shake  gently.  Separate  the  chloroform  extract  and  add 
to  it  a  drop  of  an  alcoholic  solution  of  zinc  chloride.  Note  the  rose- 
red  color  and  the  greenish  fluorescence.  If  the  solution  is  turbid  it 
may  be  rendered  clear  by  the  addition  of  a  few  c.c.  of  absolute  alcohol. 

5.  Ether-Absolute  Alcohol  Test. — Mix  urine  and  pure  ether 
in  equal  volumes  and  shake  gently  in  a  separatory  funnel.  Separate 
the  ether  extract,  evaporate  it  to  dryness,  and  dissolve  the  residue  in 
2-3  c.c.  of  absolute  alcohol.  Note  the  greenish  fluorescence.  Examine 
the  solution  spectroscopically  and  observe  the  characteristic  absorp- 
tion-band (see  Absorption  Spectra,  Plate  11). 

6.  Ring  Test. — Acidify  25  c.c.  of  urine  with  2-3  drops  of  con- 
centrated hydrochloric  acid,  add  5  c.c.  of  chloroform  and  shake  the 
mixture.  Separate  the  chloroform,  ])la(c  il  in  a  test  tube,  and  add 
carefully  3-5  c.c.  of  an  alcoholic  solution  of  zinc  acetate.  Observe 
the  formation  of  a  green  ring  at  the  zone  of  contact  of  the  two  fluids. 
If  the  tube  is  shaken  a  fluorescence  may  be  observed. 

C.  UROERYTHRIN. 

This  ];igment  is  frc'|uenlly  present  in  small  amount  in  normal 
urine.  The  red  color  of  urinary  sediments  is  due  in  great  part  to  the 
presence  of  uroerythrin.  It  is  easily  soluble  iti  amyl  ahoiioi,  slightly 
soluble  in  acetic  ether,  absolute  alcohol,  or  ( liloroforni,  and  nearly 
insoluble  in  water.  Dilute  solutions  of  uroerythrin  are  pink  in  color 
while  concentratefl  solutions  are  f)range  red  or  bright  red:  none  ol  its 


URINE.  289 

solutions  fluoresce.  Uroerythrin  is  increased  in  amount  after  strenu- 
ous physical  exercise,  digestive  disturbances,  fe\'ers,  certain  li\"er 
disorders,  and  in  various  other  pathological  conditions. 

PTOMAINES  AND  LEUCOMAINES. 

These  toxic  substances  are  said  to  be  present  in  small  amount  in 
normal  urine.  Very  little  is  known,  definitely,  however,  about  them. 
It  is  claimed  that  five  different  poisons  may  be  detected  in  the  urine, 
and  it  is  further  stated  that  each  of  these  substances  produces  a  spe- 
cific and  definite  symptom  when  injected  intravenously  into  a  rabbit. 
The  resulting  symptoms  are  narcosis,  salivation,  mydriasis,  paralysis, 
and  convulsions.  The  day  urine  is  principally  narcotic  and  is 
2-4  times  as  toxic  as  the  night  urine  which  is  chiefly  productive  of 
convulsions. 

PURINE  BASES. 

The  purine  bases  found  in  human  urine  are  adenine,  carnine, 
epiguanine,  episarkine,  guanine,  xanthine,  heteroxanthine,  hypo- 
xanthine,  paraxanthine,  and  i-methylxanthine.  The  main  bulk  of  the 
purine  base  content  of  the  urine  is  made  up  of  paraxanthine,  hetero- 
xanthine and  i-methylxanihine  which  are  derived  for  the  most  part 
from  the  caffeine,  theobromine,  and  theophylline  of  the  food.  The 
total  purine  base  content  is  made  up  of  the  products  of  two  distinct 
forms  of  metabolism,  i.  e.,  metabolism  of  ingested  nucleins  and  purines 
and  metabolism  of  tissue  nuclein  material.  Purine  bases  resulting 
from  the  first  form  of  metabolism  are  said  to  be  of  exogenous  origin 
whereas  those  resulting  from  the  second  form  of  metabolism  are  said 
to  be  of  endogenous  origin.  The  daily  output  of  purine  bases  by  the 
urine  is  extremely  small  and  varies  greatly  with  the  individual  (16-60 
milligrams).  The  output  is  increased  after  the  ingestion  of  nuclein 
material  as  well  as  after  the  increased  destruction  of  leucocytes.  A 
well  marked  increase  accompanies  leukaemia.  Edsall  has  very  recently 
shown  that  the  output  of  purine  bases  by  the  urine  is  increased  as  a 
result  of  X-ray  treatment. 

Experiment. 

I.  Formation  of  the  Silver  Salts. — Add  an  excess  of  magnesia 
mixture^  to  25  c.c.  of  urine.     Filter  off  the  precipitate  and  add  am- 

'■  Magnesia  mixture  may  be  prepared  as  follows:  Dissolve  175  grams  of  MgSO^  and 
350  grams  of  NH,C1  in  1400  c.c.  of  distilled  water.     Add  700  grams  of  concentrated 
NH^OH,  mix  very  thoroughly  and  preserve  the  mixture  in  a  glass-stoppered  bottle. 
10 


290  PHYSIOLOGICAL    CHEMISTRY, 

moniacal  silver  solution^  to  the  filtrate.  A  precipitate  composed  of 
the  silver  salts  of  the  various  purine  bases  is  produced.  The  purine 
bases  may  be  determined  quantitatively  by  means  of  Kriiger  and 
Schmidt's  method  (see  p.  399),  or  Wclkcr's  method  (see  p.  398). 

2.  Inorganic  Physiological  Constituents. 

Ammonia. 

Next  to  urea,  ammonia  is  the  most  important  of  the  nitrogenous 
end-products  of  protein  metabolism.  Ordinarily  about  2.5-4.5  per 
ce'nt  of  the  total  nitrogen  of  the  urine  is  eliminated  as  ammonia  and 
on  the  average  this  would  be  about  0.7  gram  per  day.  Under  normal 
conditions  the  ammonia  is  present  in  the  urine  in  the  form  of  the 
chloride,  phosphate,  or  sulphate.  This  is  due  to  the  fact  that  combina- 
tions of  this  sort  are  not  oxidized  in  the  organism  to  form  urea,  but  are 
excreted  as  such.  This  explains  the  increase  in  the  output  of  ammonia 
which  follows  the  administration  of  the  ammonium  salts  of  the  mineral 
acids  or  of  the  acids  themselves.  On  the  other  hand,  when  ammonium 
acetate  and  many  other  ammonium  salts  of  certain  organic  acids  are 
administered  no  increase  in  the  output  of  ammonia  occurs  since  the 
salt  is  oxidized  and  its  nitrogen  ultimately  appears  in  the  urine  as 
urea. 

The  acids  formed  during  the  process  of  protein  destruction  within 
the  body  have  an  influence  upon  the  excretion  of  ammonia  similar  to 
that  exerted  by  acids  which  have  been  administered.  Therefore  a 
pathological  increase  in  the  output  of  ammonia  is  observed  in  such 
diseases  as  are  accompanied  by  an  increased  and  imperfect  protein 
metabolism,  and  especially  in  diabetes,  in  which  disease  diacetic  acid 
and  ./J-oxybutyric  acid  are  found  in  the  urine  in  combination  with  the 
ammonia. 

As  the  result  of  recent  experiments  Folin  claims  that  a  ])ronounccd 
decrease  in  the  extent  of  ])r<)tcin  metabolism,  as  measured  by  the 
total  nitrogen  in  the  urine,  is  fre((uently  accompanied  by  a  decreased 
elimination  of  ammonia.  The  ammonia  elimination  is  therefore 
probably  determined  by  other  factors  than  the  total  protein  catabolism 
as  such.  Furthermore,  he  believes  thai  a  dec  ided  decrease  in  the 
total  nitrogen  excretion  is  always  accompanied  by  a  relative  increase 
in  the  ammonia  nitrogen,  j)rovided  the  food  is  of  a  character  yielding 
an   alkaline  ash. 

'  Ammoniai.al    silver   solution    may    Ije    prcparcfl    anordin}^    to   diici  tioiis  given    on 
page  401. 


URINE.  291 

The  quantitative  determination  of  ammonia  must  be  made  upon 
the  fresh  urine  since  upon  standing  the  normal  urine  will  undergo 
ammoniacal  fermentation  (see  page  253). 

Sulphates. 

Sulphur  in  combination  is  excreted  in  two  forms  in  the  urine; 
first,  as  loosely  combined,  tmoxidized  or  neutral  sidphur,  and,  second, 
as  oxidized  or  acid  sulphur.  The  loosely  combined  sulphur  is  excreted 
mainly  as  a  constituent  of  such  bodies  as  cystine,  cysteine,  taurine, 
hydrogen  sulphide,  ethyl  sulphide,  thiocyanates,  sulphonic  acids, 
oxyproteic  acid,  alloxyproteic  acid,  and  uroferric  acid.  The  amount 
of  loosely  combined  sulphur  eliminated  is  in  great  measure  independent 
of  the  extent  of  protein  decomposition  or  of  the  total  sulphur  excretion. 
In  this  characteristic  it  is  somewhat  similar  to  the  excretion  of  creatinine. 
The  oxidized  sulphur  is  eliminated  in  the  form  of  sulphuric  acid, 
principally  as  salts  of  sodium,  potassium,  calcium,  and  magnesium; 
a  relatively  small  amount  occurs  in  the  form  of  ethereal  sulphuric  acid, 
i.  e.,  sulphuric  acid  in  combination  with  such  aromatic  bodies  as  phenol, 
indole,  skatole,  cresol,  pyrocatechin,  and  hydroquinone.  Sulphuric 
acid  in  combination  with  Na, '  K,  Ca  or  Mg  is  sometimes  termed 
inorganic  or  preformed  sulphuric  acid,  whereas  the  ethereal  sulphuric 
acid  is  sometimes  called  conjugate  sidphuric  acid.  The  greater  part  of 
the  sulphur  is  eliminated  in  the  oxidized  form,  but  the  absolute  per- 
centage of  sulphur  excreted  as  the  preformed,  ethereal  or  loosely 
combined  type  depends  upon  the  total  quantity  of  sulphur  present, 
i.  e.,  there  is  no  definite  ratio  between  the  three  forms  of  sulphur 
which  will  apply  under  all  conditions.  The  preformed  sulphuric 
acid  may  be  precipitated  directly  from  acidified  urine  with  BaCl2, 
whereas  the  ethereal  sulphuric  acid  must  undergo  a  preliminary 
boiling  in  the  presence  of  a  mineral  acid  before  it  can  be  so  precipitated. 

The  sulphuric  acid  excreted  by  the  urine  arises  principally  from 
the  oxidation  of  protein  material  within  the  body;  a  relatively  small 
amount  is  due  to  ingested  sulphates.  Under  normal  conditions  about 
2.5  grams  of  sulphuric  acid  is  eliminated  daily.  Since  the  sulphuric 
acid  content  of  the  urine  has,  for  the  most  part,  a  protein  origin  and 
since  one  of  the  most  important  constituents  of  the  protein  molecule 
is  nitrogen,  it  would  be  reasonable  to  suppose  that  a  fairly  definite 
ratio  might  exist  between  the  excretion  of  these  two  elements.  How- 
ever, when  we  appreciate  that  the  percentage  content  of  N  and  S 
present  in  different  proteins  is  subject  to  rather  wide  variations,  the 


292 


PHYSIOLOGICAL    CHEMISTRY. 


fixing  of  a  ratio  which  will  express  the  exact  relation  existing  between 
these  two  substances,  as  they  appear  in  the  urine  as  end-products  of 
protein  metabolism,  is  practically  impossible.  It  has  been  suggested 
that  the  ratio  5  :  i  expresses  this  relation  in  a  general  way. 

Pathologically,  the  excretion  of  sulphuric  acid  by  the  urine  is  in- 
creased in  acute  fevers  and  in  all  other  diseases  marked  l^y  a  stimu- 
lated metabolism,  whereas  a  decrease  in  the  sulphuric  acid  excretion 
is  observed  in  those  diseases  which  are  accompanied  In'  a  loss  of 
appetite  and  a  diminished  metabolic  activity. 

Experiments. 

1.  Detection  of  Inorganic  Sulphuric  Acid. — Place  about  10  c.c. 
of  urine  in  a  test-tube,  acidify  with  acetic  acid  and  add  some  barium 
chloride  solution.     A  white  precipitate  of  barium  sulphate  forms. 

2.  Detection  of  Ethereal  Sulphuric  Acid. — Filter  off  the  barium 
sulphate  precipitate  formed  in  the  above  experiment,  add  i  c.c.  of 
hydrochloric  acid  and  a  little  barium  chloride  solution  to  the  filtrate 
and  heat  the  mixture  to  boiling  for  1-2  minutes.  Note  the  aj)pearance 
of  a  turbidity  due  to  the  presence  of  sulphuric  acid  which  has  been 
separated  from  the  ethereal  sulphates  and  has  combined  with  the 
barium  of  the  BaCl,  to  form  BaSO^.' 

3.  Detection  of  Loosely  Combined  or  Neutral  Sulphur. — 
Place  about   10  c.c.  (jf  urine  in  a  test-tul)e,  introduce  a  small  piece  of 

zinc,  add  sufficient  hydrochloric  acid 
to  cause  a  gentle  evolution  of  hydro- 
gen, and  over  the  mouth  of  the  tube 
place  a  filter  paper  saturated  with 
plumbic  acetate  solution.  Jn  a  short 
time  the  j)ortion  of  the  pai)er  in  con- 
tact with  the  vapors  within  the  test- 
tube  becomes  blackened  due  to  tin- 
f<jrmation  of  lead  sulphide.  The 
nascent  hydrogen  has  reacted  with  the 
loosely  combined  or  neutral  sulphur 
to  f(jrm  hydrogen  sulphide  and  this  gas  coming  in  (ontact  with  the 
jjlumbic  acetate  ]jajjer  has  caused  the  production  of  the  black  lead 
sulphide.  Sulphur  in  the  form  of  inorganic  or  ethereal  sulphuric  acid 
does  nfjt  res]>ond  to  this  test. 

4.  Calcium  Sulphate  Crystals.  Place  10  i\c.  of  urine  in  a  test- 
tube,  add  10  droj^s  (jf  calcium  chloride  solution  and  allow  the  tube  to 
stand   until   crystals   form.      Examine   the  calcium   sulphate  crystals 


J'lo.    95.— Caixixj.vi    SuM'HATK. 
{Ilensel  and  Weil.) 


URINE.  293 

under  the  microscope  and  compare  them  with  those  shown  in  Fig.  95, 
page  292. 

Chlorides. 

Next  to  urea,  the  chlorides  constitute  the  chief  solid  constituent 
of  the  urine.  The  principal  chlorides  found  in  the  urine  are  those 
of  sodium,  potassium,  ammonium,  and  magnesium,  with  sodium 
chloride  predominating.  The  excretion  of  chloride  is  dependent, 
in  great  part,  upon  the  nature  of  the  diet,  but  on  the  average  the 
daily  output  is  about  10-15  grams,  expressed  as  sodium  chloride. 
Copious  water-drinking  increases  the  output  of  chlorides  consider- 
ably. Because  of  their  solubility,  chlorides  are  never  found  in  the 
urinary  sediment. 

Since  the  amount  of  chlorides  excreted  in  the  urine  is  due  pri- 
marily to  the  chloride  content  of  the  food  ingested,  it  follows  that 
a  decrease  in  the  amount  of  ingested  chloride  will  likewise  cause  a 
decrease  in  the  chloride  content  of  the  urine.  In  cases  of  actual 
fasting  the  chloride  content  of  the  urine  may  be  decreased  to  a  slight 
trace  which  is  derived  from  the  body  fluids  and  tissues.  Under  these 
conditions,  however,  an  examination  of  the  blood  of  the  fasting  subject 
will  show  the  percentage  of  chlorides  in  this  fluid  to  be  approximately 
normal.  This  forms  a  very  striking  example  of  the  care  nature  takes 
to  maintain  the  normal  composition  of  the  blood.  There  is  a  limit  to 
the  power  of  the  body  to  maintain  this  equilibrium,  however,  and  if 
the  fasting  organism  be  subjected  to  the  influence  of  diuretics  for  a 
time,  a  point  is  reached  where  the  composition  of  the  blood  can  no 
longer  be  maintained  and  a  gradual  decrease  in  its  chloride  content 
occurs  which  finally  results  in  death.  Death  is  supposed  to  result 
not  so  much  because  of  a  lack  of  chlorine  as  from  a  deficiency  of  sodium. 
This  is  shown  from  the  fact  that  potassium  chloride,  for  instance, 
cannot  replace  the  sodium  chloride  of  the  blood  when  the  latter  is 
decreased  in  the  manner  above  stated.  When  this  substitution  is 
attempted  the  potassium  salt  is  excreted  at  once  in  the  urine,  and 
death  follows  as  above  indicated. 

Pathologically,  the  excretion  of  chlorides  may  be  decreased  in 
some  fevers,  chronic  nephritis,  croupous  pneumonia,  diarrhoea,  cer- 
tain stomach  disorders,  and  in  acute  articular  rheumatism. 

Experiment. 

Detection  of  Chlorides  in  Urine. — Place  about  5  c.c.  of  urine  in 
a  test-tube,  render  it  acid  with  nitric  acid  and  add  a  few  drops  of  a 


294  PHYSIOLOGICAL   CHEMISTRY. 

solution  of  argentic  nitrate.  A  white  precipitate,  due  to  the  formation 
of  argentic  chloride,  is  produced.  This  precipitate  is  soluble  in  am- 
monium hydroxide. 

Phosphates. 

Phosphoric  acid  exists  in  the  urine  in  two  genet"al  forms:  First, 
that  in  combination  with  the  alkali  metals,  sodium  and  potassium, 
and  the  radical  ammonium;  second,  that  in  combination  with  the 
alkaline  earths,  calcium  and  magnesium.  Phosphates  formed  through 
a  union  of  phosphoric  acid  with  the  alkali  metals  are  termed  alkaline 
phosphates,  or  phosphates  of  the  alkali  metals,  whereas  phosphates 
formed  through  a  union  of  phosphoric  acid  with  the  alkaline  earths 
are  termed  earthy  phosphates,  or  phosphates  of  the  alkaline  earths. 

Three  series  of  salts  are  formed  by  phosphoric  acid:  Normal, 
MgPO^/  mono-hydrogen,  MjHPO^,  and  di-hydrogen,  MHjPO^. 
The  di-hydrogen  salts  are  acid  in  reaction,  and  it  was  generally  be- 
lieved that  about  60  per  cent  of  the  total  phosphate  content  of  the 
urine  was  in  the  form  of  this  type  of  salt,  and  that  the  acidity  of  the 
urine  was  due  in  great  part  to  the  presence  of  sodium  di-hydrogen 
phosphate.  Recently,  however,  it  has  been  cjuite  clearly  shown  that 
the  normal  acidity  of  the  urine  is  not  due  to  the  presence  of  this  salt, 
but  is  due,  at  least  in  part,  to  the  presence  of  various  acidic  radicals. 
In  this  connection  Folin  believes  that  the  phosphates  in  clear  acid 
urine  are  all  of  the  mano -hydro gen  type,  and  that  the  acidity  of  the 
urines  of  this  character  is  generally  greater  than  the  combined  acidity 
of  all  the  phosphates  present;  the  excess  in  the  acidity  above  that  due 
to  phosphates  he  believes  to  be  due  to  free  organic  acids.  The  obser- 
vation has  recently  been  made  that  urine  may  be  separated  into  two 
portions,  one  part  consisting  almost  entirely  of  inorganic  matter 
including  practically  all  of  the  phosphates  and  having  an  alkaline 
reaction,  the  other  containing  practically  all  of  the  organic  substances 
and  no  phosphates  and  having  an  acid  reaction. 

In  bones  the  phos])h;Llcs  occur  principally  in  the  form  of  the  nor- 
mal salts  of  calcium  and  magnesium.  The  mono-hydrogen  salts  as 
a  class  are  alkaline  in  reaction  to  litmus,  and  it  is  to  the  i)resence  of 
di-sodium  hydrogen  phosphate,  NajHPO^,  ihal  \Uv  greater  ])art  of 
the  alkalinity  of  the  saliva  is  due. 

The  excretion  of  phosphoric  acid  is  extremely  variable,  but  on  the 
average  the  total  outf)ut  for  24  hours  is  about   2.5;   grams,  expressed 

'  M  may  be  occupied  by  any  of  Uic  alkali  metals  ot  all:aline  earths. 


URINE.  295 

as  PgOg.  Ordinarily  the  total  output  is  distributed  between  alkaline 
phosphates  and  earthy  phosphates  approximately  in  the  ratio  2:1. 
The  greater  part  of  this  phosphoric  acid  arises  from  the  ingested  food, 
either  from  the  preformed  phosphates  or  more  especially  from  the 
phosphorus  in  organic  combination  such  as  we  find  it  in  phospho- 
proteins,  nucleo proteins  and  lecithins;  the  phosphorus-containing  tissues 
of  the  body  also  contribute  to  the  total  output  of  this  element.  Alka- 
line phosphates  ingested  with  the  food  have  a  tendency  to  increase 
the  phosphoric  acid  content  of  the  urine  to  a  greater  extent  than  the 
earthy  phosphates  so  ingested.  This  is  due,  in  a  measure,  to  the  fact 
that  a  portion  of  the  earthy  phosphates,  under  certain  conditions,  may 
be  precipitated  in  the  intestine  and  excreted  in  the  feces;  this  is  es- 
pecially to  be  noted  in  the  case  of  herbivorous  animals.  Since  the  ex- 
tent to  which  the  phosphates  are  absorbed  in  the  intestine  depends 
upon  the  form  in  which  they  are  present  in  the  food,  under  ordinary 
conditions,  there  can  be  no  absolute  relationship  between  the  urinary 
output  of  nitrogen  and  phosphorus.  If  the  diet  is  constant,  however, 
from  day  to  day,  thus  allowing  of  the  preparation  of  both  a  nitrogen 
and  a  phosphorus  balance,^  a  definite  ratio  may  be  established.  In 
experiments  upon  dogs,  which  were  fed  an  exclusive  meat  diet,  the 
ratio  of  nitrogen  to  phosphorus,  in  the  urine  and  feces,  was  found 
to  be  8.1  : 1. 

Pathologically  the  excretion  of  phosphoric  acid  is  increased  in 
such  diseases  of  the  bones  as  diffuse  periostosis,  osteomalacia,  and 
rickets;  according  to  some  investigators,  in  the  early  stages  of  pulmon- 
ary tuberculosis,  in  acute  yellow  atrophy  of  the  liver,  in  diseases  which 
are  accompanied  by  an  extensive  decomposition  of  nervous  tissue,  and 
after  sleep  induced  by  potassium  bromide  or  chloral  hydrate  (Mendel). 
It  is  also  increased  after  copious  water-drinking.  A  decrease  in  the 
excretion  of  phosphates  is  at  times  noted  in  febrile  affections,  such  as 
the  acute  infectious  diseases;  in  pregnancy,  in  the  period  during  which 
the  foetal  bones  are  forming,  and  in  diseases  of  the  kidneys,  because 
of  non-elimination. 

Experiments. 

I.  Formation  of  '* Triple  Phosphate." — Place  some  urine  in  a 
beaker,  render  it  alkaline  with  ammonium  hydroxide,  add  a  small 
amount  of  magnesium  sulphate  solution  and  allow  the  beaker  to  stand 

*  In  metabolism  experiments,  a  statement  showing  the  relation  existing  between  the 
nitrogen  content  of  the  food  on  the  one  hand  and  that  of  the  urine  and  feces  on  the  other, 
for  a  definite  period,  is  termed  a  nitrogen  balance  or  a  "balance  of  the  income  and  outgo 
of  nitrogen." 


ig6 


PHYSIOLOGICAL    CHEMISTRY. 


in  a  cool  place  over  night.  Crystals  of  ammonium  magnesium  phos- 
phate, "triple  phosphate,'"  form  under  these  conditions.  Examine  the 
crv'stalline  sediment  under  the  microscope  and  compare  the  forms  of 
the  crystal?  with  those  shown  in  Fig.  g6,  below. 

2.  "Triple  Phosphate"  Crystals  in Ammoniacal Fermentation. 
— Stand  some  urine  aside  in  a  beaker  for  several  days.  Ammoniacal 
fermentation  will  develop  and  "triple  phosphate"  crystals  will  form. 
Examine  the  sediment  under  the  microscope  and  compare  the  crystals 
with  those  shown  in  Fig.  96,  below. 


>f 


\ 


Fig.  96. — "Triple  Phosph.\te."     {0,qden.) 


3.  Detection  of  Earthy  Phosphates.  Place  10  c.c.  of  urine  in  a 
test-tube  and  render  it  alkaline  with  ammonium  hydroxide.  Warm 
the  mixture  and  note  the  separation  of  a  y)recipitate  of  earthy  phosphates. 

4.  Detection  of  Alkaline  Phosphates. — Filter  off  the  earthy 
phosphates  as  formed  in  the  last  experiment,  and  add  a  small  amount 
of  magnesia  mixture  (see  page  289)  to  the  filtrate.  Now  warm  the 
mixture  and  observe  the  formation  of  a  white  y)recipitate  due  to  the 
presence  of  alkaline  jjhosphates.  Nolc  the  (liffcrencc  in  the  size  of 
the  [)recii)ilates  of  the  two  forms  of  phos])hates  from  this  same 
volume  of  urine.  Which  form  of  ph(;s])hatcs  was  present  in  the 
larger  amount,  earthy  or  alkaline? 

V  Influence  upon  Fehling's  Solution. — Place  2  c.c.  of  Fehling's 
solution  in  a  lesl-lube,  dilute  it  with  4  volumes  of  water  and  heat  to 
boiling.  Add  a  solution  of  sodium  dihydrogen  j)hos};hate,  NallJH)^, 
a  small  amount  at  a  time,  and  heat  after  each  addition.  What  do  you 
observe?  What  does  this  observation  force  you  to  conclude  regarding 
the  interference  of  jjhosjjhates  in  the  testing  of  diabetir  urine  by  means 
of  Fehling's  test? 


URINE.  297 

Sodium  and  Potassium. 

The  elements  sodium  and  potassium  are  always  present  in  the  urine. 
Usually  they  are  combined  with  such  acidic  radicals  as  CI,  CO3,  SO^ 
and  PO4.  The  amount  of  potassium,  expressed  as  K^O,  excreted  in 
24  hours  by  an  adult,  subsisting  upon  a  mixed  diet,  is  on  the  average 
2-3  grams,  whereas  the  amount  of  sodium,  expressed  as  NajO,  under 
the  same  conditions,  is  ordinarily  4-6  grams.  The  ratio  of  K  to  Na  is 
generally  about  3:5.  The  absolute  quantity  of  these  elements  excreted 
depends,  of  course,  in  large  measure,  upon  the  nature  of  the  diet. 
Because  of  the  non-ingestion  of  NaCl  and  the  accompanying  destruc- 
tion of  potassium-containing  body  tissues,  the  urine  during  fasting 
contains  more  potassium  salts  than  sodium  salts. 

Pathologically  the  output  of  potassium,  in  its  relation  to  sodium, 
may  be  increased  during  fever;  following  the  crisis,  however,  the  out- 
put of  this  element  may  be  decreased.  It  may  also  be  increased  in 
conditions  associated  with  acid  intoxication. 

Calcium  and  Magnesiimi. 

The  greater  part  of  the  calcium  and  magnesium  excreted  in  the 
urine  is  in  the  form  of  phosphates.  The  daily  output,  which  depends 
principally  upon  the  nature  of  the  diet,  aggregates  on  the  average  about 
I  gram  and  is  made  up  of  the  phosphates  of  calcium  and  magnesium 
in  the  proportion  of  i  :2.  The  percentage  of  calcium  salts  present  in 
the  urine  at  any  one  time  forms  no  dependable  index  as  to  the  absorp- 
tion of  this  class  of  salts,  since  they  are  again  excreted  into  the  intestine 
after  absorption.  It  is  therefore  impossible  to  draw  any  satisfactory 
conclusions  regarding  the  excretion  of  the  alkaline  earths  unless  we 
obtain  accurate  analytical  data  from  boLh  the  feces  and  the  urine. 

\'ery  little  is  known  positively  regarding  the  actual  course  of  the 
excretion  of  the  alkaline  earths  under  pathological  conditions  except 
that  an  excess  of  calcium  is  found  in  acid  intoxication  and  some  diseases 
of  the  bones. 

Carbonates. 

Carbonates  generally  occur  in  small  amount  in  the  urine  of  man 
and  carnivora  under  normal  conditions,  whereas  much  larger  quanti- 
ties are  ordinarily  present  in  the  urine  of  herbivora.  The  alkaline 
reaction  of  the  urine  of  herbivora  is  dependable  in  great  measure  upon 
the  presence  of  carbonates.     In  general  a  urine  containing  carbonates 


298  PHYSIOLOGICAL   CHEMISTRY. 

in  appreciable  amount  is  turbid  when  passed  or  becomes  so  shortly- 
after.  These  bodies  ordinarily  occur  as  alkali  or  alkaline  earth  com- 
pounds and  the  turbid  character  of  urine  containing  them  is  usually 
due  principally  to  the  latter  class  of  substances.  The  carbonates  of 
the  alkaline  earths  are  often  found  in  amorphous  urinary  sediments. 

Iron. 

Iron  is  present  in  small  amount  in  normal  urine.  It  probably 
occurs  partly  in  inorganic  and  partly  in  organic  combination.  The 
iron  contained  in  urinary  pigments  or  chromogens  is  in  organic  com- 
bination. According  to  different  investigators  the  iron  content  of 
normal  urine  will  probably  not  average  more  than  0.00 1  gram  per  day. 

Experiment. 

Detection  of  Iron  in  Urine. — Evaporate  a  convenient  volume 
(ia-15  c.c.)  of  urine  to  dryness.  Incinerate  and  dissolve  the  residue 
in  a  few  drops  of  iron-free  hydrochloric  acid  and  dilute  the  acid  solu- 
tion with  5  c.c.  of  water.  Divide  the  acid  solution  into  two  parts  and 
make  the  following  tests:  (a)  To  the  first  part  add  a  solution  of  ammo- 
nium thiocyanate;  a  red  color  indicates  the  presence  of  iron,  {b)  To 
the  second  part  of  the  solution  add  a  little  potassium  ferrocyanide  solu- 
tion;  a  precipitate  of  Prussian  blue  forms  upon  standing. 

Fluorides,  Nitrates,  Silicates  and  Hydrogen  Peroxide. 

These  substances  are  all  found  in  traces  in  human  urine  under 
normal  conditions.  Nitrates  are  undoubtedly  introduced  into  the 
organism  in  the  water  and  ingested  food.  The  average  excretion  of 
nitrates  is  about  0.5  gram  per  day,  the  output  being  the  largest  upon  a 
vegetable  diet  and  smallest  upon  a  meat  diet.  Nitrates  are  found  only 
in  urine  which  is  undergoing  decomposition  and  are  formed  from 
nitrates  in  the  course  of  ammoniacal  fermentation.  Hydrogen  per- 
oxide has  been  detected  in  the  urine,  but  its  presence  is  ])elieved  to  pos- 
sess no  pathological  importance. 


CHAPTER  XIX. 
URINE:  PATHOLOGICAL  CONSTITUENTS/ 


Dextrose. 


Proteins 


Serum  albumin. 
Serum  globulin. 

Deutero-proteose. 
Proteoses  j  Hetero-proteose. 

"Bence- Jones'  protein." 


Blood 


Peptone. 

Nucleoprotein. 

Fibrin. 

Oxy  haemoglobin. 
Form  elements. 
Pigment. 


Bile 


Pigments. 

Acids. 
Acetone. 
Diacetic  acid. 
^-Oxybutyric  acid. 
Conjugate  glycuronates. 
Pentoses. 
Fat. 

Haematoporphyrin. 
Lactose. 
Galactose. 
Lsevulose. 
Inosite. 
Laiose. 
Melanin. 
Urorosein. 
Unknown  substances. 

DEXTROSE. 

Traces  of  this  sugar  occur  in  normal  urine,  but  the  amount  is  not 
sufificient  to  be  readily  detected  by  the  ordinary  simple  qualitative 


'■  See  note  at  the  bottom  of  page  259. 


299 


300  PHYSIOLOGICAL   CHEMISTRY. 

tests.  There  are  two  distinct  types  of  pathological  glycosuria,  /.  c, 
transitory  glycosuria  and  persistent  glycosuria.  The  transitory  type 
may  follow  the  ingestion  of  an  excess  of  sugar,  causing  the  assimilation 
limit  to  be  exceeded,  or  it  may  accompany  any  one  of  several  disorders 
which  cause  impairment  of  the  power  of  assimilating  sugar.  In  the 
persistent  type  large  amounts  of  sugar  are  excreted  daily  in  the  urine 
for  long  periods  of  time.  Under  such  circumstances  a  condition  known 
as  diabetes  mellitus  exists.  Ordinarily,  diabetic  urine  which  contains 
a  high  percentage  of  sugar  possesses  a  faint  yellow  color,  a  high  specific 
gravity,  and  a  volume  which  is  above  normal. 

Experiments. 

I.  Phenylhydrazine  Reaction. — Test  the  urine  according  to  one 
of  the  following  methods:  (a)  To  a  small  amount  of  phenylhydrazine 
mixture,  furnished  by  the  instructor/  add  5  c.c.  of  the  urine,  shake 
well,  and  heat  on  a  boiling  water-bath  for  one-half  to  three-quarters  of 
an  hour.  Allow  the  tube  to  cool  slowly  and  examine  the  crystals  micro- 
scopically (Plate  III,  opposite  page  23).  If  the  solution  has  become 
too  concentrated  in  the  boiling  process  it  will  be  light-red  in  color  and 
no  crystals  will  separate  until  it  is  diluted  with  water. 

Yellow  crystalline  bodies  called  osazones  are  formed  from  certain 
sugars  under  these  conditions,  in  general  each  individual  sugar  giving 
rise  to  an  osazone  of  a  definite  crystalline  form  which  is  typical  for  that 
sugar.  It  is  important  to  remember  in  this  connection  that,  of  the 
simple  sugars  of  interest  in  [)hysiological  chemistry,  dextrose  and  Isevu- 
lose  yield  the  same  osazone,  with  phenylhydrazine.  Each  osazone  has 
a  definite  melting-point,  and  as  a  further  and  more  accurate  means  of 
identification  it  may  be  recrystallized  and  identified  by  the  determina- 
tion of  its  melting-j)oint  and  nitrogen  content.  The  reaction  taking 
place  in  the  formation  of  phenyldextrosazone  is  as  follows: 

C.,H,/J„^2(H3N.NH.C„H,)  =  C„H,„(),(N.NH.C„Hj,+  2H/)-|-H2. 

iJexirijsc,  Phcnylhyilr;izinc.  Phenyl'lextrosfizone. 

ib)  Place  5  c.c.  of  the  urine  in  a  test  tube,  add  1  c.c.  of  plun\'lliy- 
drazine-acetate  scjhition  furnished  by  \.hv  instructor,"  and  heal  on  a 
boiling  water-bath  for  fjne-half  to  three  (juarters  of  an  hour.  AHow 
the  li()uid  to  cool  slowly  and  examine  the  crystals  microscopically 
(Plate  111.  opposite  \).  23). 

'  This  mixture  is  propared  Ijy  comljining  one  part  of  |jlienylliy(lrazinc-hy(lro<hloride 
and  two  parts  o'  s<jdium  acetate,  \)y  weight.     These  arc  thorou^lily  mixed  in  a  mortar. 

'■'  This  solution  is  preparer!  by  mixing;  one  part  hy  volume,  in  each  case,  of  j^hu  ial 
acetic  arid,  one  part  of  water  and  two  parts  of  phenylhydrazine  (the  base). 


URINE.  301 

The  phenylhydrazine  test  has  been  so  modified  by  CipolHna  as  to 
be  of  use  as  a  rapid  clinical  test.  The  directions  for  this  test  are  given 
in  the  next  experiment. 

2.  CipoUina's  Test. — Thoroughly  mix  4  c.c.  of  urine,  5  drops  of 
phenylhydrazine  (the  base)  and  1/2  c.c.  of  glacial  acetic  acid  in 
a  test-tube.  Heat  the  mixture  for  about  one  minute  over  a  low  flame, 
shaking  the  tube  continually  to  prevent  loss  of  fluid  by  bumping.  Add 
4-5  drops  of  potassium  hydroxide  or  sodium  hydroxide  (sp.  gr.  1.16), 
being  certain  that  the  fluid  in  the  test-tube  remains  acid;  heat  the  mix- 
ture again  for  a  moment  and  then  cool  the  contents  of  the  tube.  Ordi- 
narily the  crystals  form  at  once,  especially  if  the  urine  possesses  a  low 
specific  gravity.  If  they  do  not  appear  immediately  allow  the  tube  to 
stand  at  least  20  minutes  before  deciding  upon  the  absence  of  sugar. 

Examine  the  crystals  under  the  microscope  and  compare  them 
with  those  shown  in  Plate  III,  opposite  page  23. 

3.  Riegler's  Reaction/ — Introduce  o.i  gram  of  phenylhydrazine- 
hydrochloride  and  0.25  gram  of  sodium  acetate  into  a  test-tube,  add 
20  drops  of  the  urine  under  examination,  and  heat  the  mixture  to 
boiling.  Now  introduce  10  c.c.  of  a  3  per  cent  solution  of  potassium 
hydroxide  and  gently  shake  the  tube  and  contents.  If  the  urine  under 
examination  contains  dextrose  the  liquid  in  the  tube  will  assume  a 
red  color.  One  per  cent  dextrose  yields  an  immediate  color  whereas 
0.05  per  cent  yields  the  color  only  after  the  lapse  of  a  period  of  one- 
half  hour  from  the  time  the  alkali  is  added.  If  the  color  appears  after 
the  30-minute  interval  the  color  change  is  without  significance  inas- 
much as  sugar-free  urines  will  respond  thus.  The  reaction  is  given 
by  all  aldehydes  and  therefore  the  test  cannot  be  safely  employed  in 
testing  urines  preserved  by  formaldehyde.  Albumin  does  not  interfere 
with  the  test. 

4.  Bottu's  Test.^ — To  8  c.c.  of  Bottu's  reagent^  in  a  test-tube  add 
I  c.c.  of  the  urine  under  examination  and  mix  the  liquids  by  gentle 
shaking.     Now  heat  the  upper  portion  of  the  mixture  to  boiling,  add 

■  an  additional  1  c.c.  of  urine  and  heat  the  mixture  again  immediately. 
The  appearance  of  a  blue  color  accompanied  by  the  precipitation  of 
small  particles  of  indigo  blue  indicates  the  presence  of  dextrose  in  the 
urine  under  examination.  The  test  will  serve  to  detect  the  presence 
of  0.1  per  cent  of  dextrose  and  is  uninfluenced  by  creatinine  or  by 
ammonium  salts. 

'  Riegler:     Compt.  rend.  soc.  biol.,  66,  p.  795. 
-  Bottu:     Compt.  rend.  soc.  biol.,  66,  p.  972. 

^  This  reagent  contains  3.5  grams  of  o-nitrophen3'lpropiolic  acid  and  5  c.c.  of  a 
freshly  prepared  10  per  cent,  solution  of  sodium  hydroxide  per  liter. 


302  PHYSIOLOGICAL   CHEMISTRY. 

5.  Reduction  Tests. — To  their  aldehyde  or  ketone  structure  many 
sugars  owe  the  property  of  readily  reducing  the  alkaline  solutions  of  the 
oxides  of  metals  like  copper,  bismuth,  and  mercury;  they  also  possess 
the  property  of  reducing  ammoniacal  silver  solutions  with  the  separa- 
tion of  metallic  silver.  Upon  this  property  of  reduction  the  most 
widely  used  tests  for  sugars  are  based.  When  whitish-blue  cupric  hy- 
droxide in  suspension  in  an  alkaline  liquid  is  heated  it  is  converted  into 
insoluble  black  cupric  oxide,  but  if  a  reducing  agent  like  certain  sugars 
be  present  the  cupric  hydroxide  is  reduced  to  insoluble  yellow  cuprous 
hydroxide,  which  in  turn  on  further  heating  may  be  converted  into 
brownish-red  or  red  cuprous  oxide.  These  changes  are  indicated  as 
follows : 

OH 

/ 
Cu  —      Cu^O+H^O. 

\  Cupric  oxide 


OH 

Cupric  hydroxide 
(whitish-blue). 


(black). 


OH 

/ 
Cu 

\ 
OH 


2Cu-OH-fH20  +  0. 

OH        t 

/ 

Cu 


r^TJ  Cuprous  hydroxide 

^^  (yellow). 


OH 

Cu— OH 

I 

Cu-OH 


Cu 

\ 

b-i-H„o. 

/ 

Cu 


Cuprous  hydroxide      Cuprous  oxide 
(yellow).  (brownish-red). 

The  chemical  equations  here  discussed  are  exemplified  in  Trommer's 
and  Fehling's  tests. 

(a)  Trommer\s  Test. — To  5  c.c.  of  urine  in  a  test-tube  add  one- 
half  its  volume  of  KOH  or  NaOH.  Mix  thoroughly  and  add,  drop  by 
drop,  agitating  after  the  addition  of  each  drop,  a  very  dilute  solution  of 
cuj>ric  suljjhate.     Continue  the  addition  until  there  is  a  slight  pcrma- 


URINE.  303 

nent  precipitate  of  cupric  hydroxide  and  in  consequence  the  solution  is 
slightly  turbid.  Heat,  and  the  cupric  hydroxide  is  reduced  to  yellow 
cuprous  hydroxide  or  to  brownish-red  cuprous  oxide.  If  the  solution 
of  cupric  sulphate  used  is  too  strong,  a  small  brownish-red  precipitate 
produced  in  the  presence  of  a  low  percentage  of  dextrose  may  be  entirely 
masked.  On  the  other  hand,  if  too  httle  cupric  sulphate  is  used  a  light- 
colored  precipitate  formed  by  uric  acid  and  purine  bases  may  obscure 
the  brownish-red  precipitate  of  cuprous  oxide.  The  action  of  KOH 
or  NaOH  in  the  presence  of  an  excess  of  sugar  and  insufficient  copper 
will  produce  a  brownish  color.  Phosphates  of  the  alkaline  earths  may 
also  be  precipitated  in  the  alkaline  solution  and  be  mistaken  for  cuprous 
hydroxide.     Trommer's  test  is  not  very  satisfactory. 

(b)  Fehling's  Test. — To  about  i  c.c.  of  Fehling's  solution^  in  a  test- 
tube  add  about  4  c.c.  of  water,  and  boil.  This  is  done  to  determine 
whether  the  solution  will  of  itself  cause  the  formation  of  a  precipitate 
of  brownish-red  cuprous  oxide.  If  such  a  precipitate  forms,  the 
Fehling's  solution  must  not  be  used.  Add  urine  to  the  warm  Fehling's 
solution,  a  few  drops  at  a  time,  and  heat  the  mixture  after  each  addition. 
The  production  of  yellow  cuprous  hydroxide  or  brownish-red  cuprous 
oxide  indicates  that  reduction  has  taken  place.  The  yellow  precipi- 
tate is  more  likely  to  occur  if  the  urine  is  added  rapidly  and  in  large 
amount,  whereas  with  a  less  rapid  addition  of  smaller  amounts  of  urine 
the  brownish-red  precipitate  is  generally  formed. 

This  is  a  much  more  satisfactory  test  than  Trommer's,  but  even 
this  test  is  not  entirely  reliable  when  used  to  detect  sugar  in  the  urine. 
Such  bodies  as  conjugate  glycuronates,  uric  acid,  nucleo protein,  and  honio- 
gentisic  acid,  when  present  in  sufficient  amount,  may  produce  a  result 
similar  to  that  produced  by  sugar.  Phosphates  of  the  alkaline  earths 
may  be  precipitated  by  the  alkali  of  the  Fehling's  solution  and  in  appear- 
ance rhay  be  mistaken  for  the  cuprous  hydroxide.  Cupric  hydroxide 
may  also  be  reduced  to  cuprous  oxide  and  this  in  turn  be  dissolved  by 
creatinine,  a  normal  urinary  constituent.  This  will  give  the  urine 
under  examination  a  greenish  tinge  and  may  obscure  the  sugar  reaction 
even  when  a  considerable  amount  of  sugar  is  present, 

(c)  Benedict's  Modifications  of  Fehling' s  Test. — First  Modification. — 

^  Fehling's  solution  is  composed  of  two  definite  solutions — a  cupric  sulphate  solution 
and  an  alkaline  tartrate  solution,  which  may  be  prepared  as  follows: 

Cupric  sulphate  solution  =  ^,^.6$  grams  of  cupric  sulphate  dissolved  in  water  and  made 
up  to  500  c.c. 

Alkaline  tartrate  solution  =  12$  grams  of  potassium  hydroxide  and  173  grams  of 
Rochelle  salt  dissolved  in  water  and  made  up  to  500  c.c. 

These  solutions  should  be  preserved  separatel)'  in  rubber-stoppered  bottles  and  mixed 
in  equal  volumes  when  needed  for  use.     This  is  done  to  prevent  deterioration. 


304  PHYSIOLOGICAL    CHEMISTRY. 

To  2  c.c.  of  Benedict's  solution^  in  a  test-tube  add  6  c.c.  of  distilled 
water  and  7-9  drops  (not  more)  of  the  urine  under  examination.  Boil 
the  mixture  vigorously  for  about  15-30  seconds  and  permit  it  to  cool 
to  room  temperature  spontaneously.  (If  desired  this  process  may  be 
repeated,  although  it  is  ordinarily  unnecessary.)  If  sugar  is  present 
in  the  solution  a  precipitate  will  form  which  is  often  bhiish-grecn  or 
green  at  first,  especially  if  the  percentage  of  sugar  is  low,  and  which 
usually  becomes  yelloivish  upon  standing.  If  the  sugar  present  exceeds 
0.06  per  cent  this  precipitate  generally  forms  at  or  below  the  boiling- 
point,  whereas  if  less  than  0.06  per  cent  of  sugar  is  present  the  precipi- 
tate forms  more  slowly  and  generally  only  after  the  solution  has  cooled. 
The  greenish  precipitate  obtained  with  urines  containing  small  amounts 
of  sugar  may  be  a  compound  of  copper  with  the  sugar  or  a  compound 
of  some  constituent  of  the  urine  with  reduced  copper  oxide  instead  of 
being  a  precipitate  of  cuprous  hydroxide  or  oxide  as  is  the  case  when 
the  original  Fehling  solution  is  reduced. 

Benedict  claims  that,  whereas  the  original  Fehling  test  will  not 
serve  to  detect  sugar  when  present  in  a  concentration  of  less  than  o.i 
per  cent,  that  the  above  modification  will  serve  to  detect  sugar  when 
present  in  as  small  quantity  as  0.015-0.02  per  cent.  The  modified 
solution  used  in  the  above  test  differs  from  the  original  in  that  too 
grams  of  sodium  carbonate  is  substituted  for  the  125  grams  of  potas- 
sium hydroxide  ordinarily  used,  thus  forming  a  Fehling  solution  which 
is  considerably  less  alkaline  than  the  original.  This  alteration  in  the 
composition  of  the  Fehling  solution  is  of  advantage  in  the  detection  of 
sugar  in  the  urine  inasmuch  as  the  strong  alkalinity  of  the  ordinary 
Fehling  solution  has  a  tendency,  when  the  reagent  is  boiled  with  a 
urine  containing  a  small  amount  of  dextrose,  to  decompose  sufficient 
of  the  sugar  to  render  the  detection  of  the  remaining  portion  exceed- 
ingly dilTicult  by  the  usual  technicjue.  Benedict  claims  that  for  this 
reason  the  use  oi  his  modilicd  sohilion  [HTmits  the  (k'teclion  of  smaller 
amounts  of  sugar  than  does  the  use  of  the  ordinary  Fehling  solution. 
Benedict  has  further  modified  his  solution  for  use  in  the  ([uantilativc 
determination  of  sugar  (see  page  363). 

Second   .\ff>(/ //i(  (i/ion.''^- -Wcry   recently    l)rnc(li(  I    has   furl  her   modi 

'  Uencdict's  modii'icd  J'chling  sf)lution  consists  of  two  (iefinito  solutifnis— a  (  ui)ri(: 
.sulphate  solution  anrl  an  alkaline  tartrate  solution,  which  may  he  prcparcil  as  follows: 

C'upric  sulphate  solution  -  },^  .()^  grams  of  (:ui)ri(  suljihalc  ijissolvcd  in  walcr  and  made 
up  to  500  c.c. 

Alkaline  tartrate  soluli(m  =  too  grams  of  anhydrous  sodinm  laihonalc  ami  17-;  grams 
of  Kochelle  salt  dissolved  in  water  and  made  up  to  500  c.i  . 

'I'hese  s<jlutions  should  be  [jreserved  separately  in  rul)l)cr-stiip|i(icd  lioiilcs  -.uvl  mixed 
in  equal  volumes  when  needed  for  use.     This  is  done  to  prcvcnl  dclcrioiaiinn 

^  I'rivale  <  ommunif  alioti  from    l)r.  S.   K.   Henedi(  1. 


URINE.  305 

fied  his  solution  and  has  succeeded  in  obtaining  one  which  does  not 
deteriorate  upon  long  standing/  The  following  is  the  procedure 
for  the  detection  of  dextrose  in  the  urine:  To  5  c.c.  of  the  reagent 
in  a  test-tube  add  eight  (not  more)  drops  of  the  urine  to  be  ex- 
amined. The  fluid  is  then  boiled  vigorously  for  from  one  to  two 
minutes  and  then  allowed  to  cool  spontaneously.  In  the  presence  of  dex- 
trose the  entire  body  of  the  solution  will  he  filled  with  a  precipitate,  which 
may  be  red,  yellow,  or  green  in  color,  depending  upon  the  amount  of 
sugar  present.  If  no  dextrose  is  present,  the  solution  will  either  remain 
perfectly  clear,  or  will  show  a  very  faint  turbidity,  due  to  precipitated 
urates.  Even  very  small  quantities  of  dextrose  in  urine  (o.i  per  cent) 
yield  precipitates  of  surprising  bulk  with  this  reagent,  and  the  positive 
reaction  for  dextrose  is  the  filling  of  the  entire  body  of  the  solution 
with  a  precipitate,  so  that  the  solution  becomes  opaque.  Since  amount 
rather  than  color  of  the  precipitate  is  made  the  basis  of  this  test,  it  may 
be  applied,  even  for  the  detection  of  small  quantities  of  dextrose,  as 
readily  in  artificial  light  as  in  daylight. 

{d)  Allen's  Modification  ofFehling's  Test. — The  following  procedure 
is  recommended:  "From  7  to  8  c.c.  of  the  sample  of  urine  to  be  tested 
is  heated  to  boiling  in  a  test-tube,  and,  without  separating  any  precipi- 
tate of  albumin  which  may  be  produced,  5  c.c.  of  the  solution  of  cu- 
pric  sulphate  used  for  preparing  Fehling's  solution  is  added.  This  pro- 
duces a  precipitate  containing  uric  acid,  xanthine,  hypoxan thine,  phos- 
phates, etc.  To  render  the  precipitation  complete,  however,  it  is 
desirable  to  add  to  the  Hquid,  when  partially  cooled,  from  i  to  2  c.c.  of 
a  saturated  solution  of  sodium  acetate  having  a  feebly  acid  reaction  to 
litmus.^  The  Hquid  is  filtered  and  to  the  filtrate,  which  will  have  a 
bluish-green  color,  5  c.c.  of  the  alkaline  tartrate  mixture  used  for  pre- 
paring Fehling's  solution  is  added,  and  the  liquid  boiled  for  15-20 
seconds.     In  the  presence  of  more  than  0.25  per  cent  of  sugar,  separa- 

*  Benedict's  new  solution  has  the  following  composition: 

Cupric  sulphate    i?  -3  S'-^'^- 

Sodium  citrate    ■ 173  -o  gm. 

Sodium  carbonate  (anhydrous) 100. o  gm. 

Distilled  water  to 1000. o  c.c. 

■  With  the  aid  of  heat  dissolve  the  sodium  citrate  and  carbonate  in  about  600  c.c.  of 
water.  Pour  (through  a  folded  filter  if  necessary)  into  a  glass  graduate  and  make  up  to 
850  c.c.  Dissolve  the  cupric  sulphate  in  about  100  c.c.  of  water  and  make  up  to  150  c.c. 
Pour  the  carbonate-citrate  solution  into  a  large  beaker  or  casserole  and  add  the  cupric 
sulphate  solution  slowly,  with  constant  stirring.  The  mixed  solution  is  ready  for  use,  and 
does  not  deteriorate  upon  long  standing. 

-  Sufficient  acetic  acid  should  be  added  to  the  sodium  acetate  solution  to  render  it 
feebly  acid  to  litmus.  A  saturated  solution  of  sodium  acetate  keeps  well,  but  weaker 
solutions  are  apt  to  become  mouldy,  and  then  possess  the  power  of  reducing  Fehling's 
solution.  Hence  it  is  essential  in  all  cases  of  importance  to  make  a  blank  test  by  mi.xing 
equal  measures  of  cupric  sulphate  solution,  alkaline  tartrate  solution  and  water,  adding  a 
little  sodium  acetate  solution,  and  heating  the  mixture  to  boiUng. 


20 


3o6  PHYSIOLOGICAL   CHEMISTRY. 

tion  of  cuprous  oxide  occurs  before  the  boiling-point  is  reached;  but 
with  smaller  quantities  precipitation  takes  place  during  the  cooling  of 
the  solution,  which  becomes  greenish,  opaque,  and  suddenly  deposits 
cuprous  oxide  as  a  fine  brow^nish-red  precipitate." 

{e)  Boettger's  Test. — To  5  c.c.  of  urine  in  a  test-tube  add  i  c.c.  of 
KOH  or  NaOH  and  a  very  small  amount  of  bismuth  subnitrate,  and 
boil.  The  solution  will  gradually  darken  and  finally  assume  a  black 
color  due  to  reduced  bismuth.  If  the  test  is  made  with  urine  contain- 
ing albumin  this  must  be  removed,  by  boiling  and  filtering,  before 
applying  the  test,  since  with  albumin  a  similar  change  of  color  is  pro- 
duced (bismuth  sulphide). 

(/)  Nylander's  Test  {Almen's  Test). — To  5  c.c.  of  urine  in  a  test- 
tube  add  one-tenth  its  volume  of  Nylander's  reagent^  and  heat  for  five 
minutes  in  a  boiling  water-bath.^  The  mixture  will  darken  if  reducing 
sugar  is  present  and  upon  standing  for  a  few  moments  a  black  color 
will  appear.  This  color  is  due  to  the  precipitation  of  bismuth.  If  the 
test  is  made  on  urine  containing  albumin  this  must  be  removed,  by 
boiling  and  filtering,  before  applying  the  test,  since  with  albumin  a 
similar  change  of  color  is  produced.  Dextrose  when  present  to  the 
extent  of  0.08  per  cent  may  be  detected  by  this  reaction.  It  is  claimed 
by  Bechold  that  Nylander's  and  Boettger's  tests  give  a  negative  reaction 
with  solutions  containing  sugar  when  mercuric  chloride  or  chloroform 
is  present.  Other  observers'  have  failed  to  verify  the  inhibitory  action 
of  the  mercuric  chloride  and  have  shown  that  the  inhibitory  influence 
of  chloroform  may  be  overcome  by  raising  the  temperature  of  the  urine 
to  the  boiling-point  for  a  period  of  five  minutes  previous  to  making 
the  test. 

Urines  rich  in  indican,  uroerylhrin,  urochrome  or  hcpmatopor phyrin, 
as  well  as  urines  excreted  after  the  ingestion  of  large  amounts  of  certain 
medicinal  substances,  may  give  a  darkening  of  Nylander's  reagent  similar 
t(;  that  of  a  true  sugar  reaction.  It  is  a  disputed  point  whether  the 
urine  after  the  administration  of  urotropin  will  reduce  Nylander's 
reagent.* 

According  to  Riistin  and  OUo  ihc  addition  of  IMCl,  increases  the 

'  Nylander's  reagent  is  prcj)arc<l  hy  dif^esting  2  ^;nims  of  bismuth  sulmitrato  anrj  4 
grams  of  Rochcllc  salt  in  100  c.c .  of  a  10  per  ( cnl  potassium  hydroxidr  solution.  Tin: 
reagent  is  then  cooled  and  filtered. 

^  Hammarsten  suggests  that  the  solution  Kc  lioilcd  for  2  s  minutes  (lu  cording'  to  the 
sugar  content)  over  a  free  (lame  and  the  tulu-  ilicn  pcrmillcd  to  stand  live  mimitcs  jjefoio 
drawing  conclusions. 

M<ehfuss  and  Hawk:  Jour.  Biol.  Chem.,  VII,  p.  207,  i(,io;  also  /cidlitz:  Upsala 
Lakdreforen  Fork.,  N.  F.,  XI,  iyo6. 

*  Abt:  Archives  0/  Pediatrics,  X.XIV,  p.  275,  1907;  also  VVcilhni  hi :  Srhweiz.  Woch., 
XLVII,  p.  577,  1909. 


UEINE.  307 

delicacy  of  Nylander's  reaction.  They  claim  that  this  procedure 
causes  the  sugar  to  be  converted  quantitatively.  No  quantitative 
method  has  yet  been  devised,  however,  based  upon  this  principle. 

A  positive  Nylander  or  Boettger  test  is  probably  due  to  the  following 
reactions : 

{a)  Bi(OH)2(NO)3  +  KOH  =  Bi(OH)3  +  KN03. 

{h)  2Bi(OHJ3-30  =  Bi3  +  3H30. 

Bohmansson,^  before  testing  the  urine  under  examination  treats  it 
(10  c.c.)  with  1/5  volume  of  25  per  cent  hydrochloric  acid  and  1/2 
volume  of  bone  black.  This  mixture  is  shaken  one  minute,  then 
filtered,  and  the  neutralized  filtrate  tested  by  Nylander's  reaction. 
Bahmansson  claims  that  this  procedure  removes  certain  interfering 
substances,  notably  urochrome. 

6.  Fermentation  Test. — Rub  up  in  a  mortar  about  15  c.c.  of 
the  urine  with  a  small  piece  of  compressed  yeast.  Transfer  the  mix- 
ture to  a  saccharometer  (Fig.  2,  p.  31)  and  stand  it  aside  in  a  warm 
place  for  about  12  hours.  If  dextrose  is  present,  alcoholic  fermenta- 
tion will  occur  and  carbon  dioxide  will  collect  as  a  gas  in  the  upper 
portion  of  the  tube.  On  the  completion  of  fermentation  introduce, 
by  means  of  a  bent  pipette,  a  little  KOH  solution  into  the  graduated 
portion,  place  the  thumb  tightly  over  the  opening  in  the  apparatus  and 
invert  the  saccharometer.     Explain  the  result. 

7.  Barfoed's  Test. — Place  about  5  c.c.  of  Barfoed's  solution^  in 
a  test-tube  and  heat  to  boiling.  Add  the  urine  under  examination 
slowly,  a  few  drops  at  a  time,  heating  after  each  addition.  Reduc- 
tion is  indicated  by  the  production  of  a  red  precipitate.  If  the  pre- 
cipitate does  not  form  upon  continued  boiling  allow  the  tube  to  stand 
a  few  minutes  and  examine.     NaCl  interferes  with  this  test  (Welker). 

Barfoed's  test  is  not  a  specific  test  for  dextrose  as  is  frequently 
stated,  but  simply  serves  to  detect  monosaccharides.  Disaccharides 
will  also  respond  to  the  test,  according  to  Hinkel  and  Sherman,  if  the 
solution  is  boiled  sufficiently  long  in  contact  with  the  reagent  to  hydrolyze 
the  disaccharide  through  the  action  of  the  acetic  acid  present  in  the 
Barfoed's  solution. 

8.  Polariscopic  Examination. — For  directions  as  to  the  use  of 
the  polariscope  see  page  31. 

'  Bohmansson:     Biochem.  Zeit.,  19,  p.  281. 

2  Barfoed's  solution  is  prepared  as  follows:  Dissolve  4.  5  grams  of  neutral,  crs'stallized 
cupric  acetate  in  100  c.c.  of  water  and  add  i  .2  c.c.  of  50  per  cent  acetic  acid. 


3o8  PHYSIOLOGICAL    CHEMISTRY. 

PROTEINS. 

Normal  urine  coniains  a  trace  of  protein  material  but  the  amount 
present  is  so  slight  as  to  escape  detection  by  any  of  the  simple  tests 
in  general  use  for  the  detection  of  protein  urinary  constituents.  The 
following  are  the  more  important  forms  of  protein  material  which 
have  been  detected  in  the  urine  under  pathological  conditions: 

(i)  Serum  albumin. 
(2)  Serum  globulin. 

'  Deutero-proteose. 
(3)  Proteoses  -j  Hetero-proteose. 

"  Bence- Jones'  protein." 

(4)  Peptone. 

(5)  Nucleoprotein. 

(6)  Fibrin. 

(7)  Oxyhaemoglobin. 

ALBUMIN. 

Albuminuria  is  a  condition  in  which  serum  albumin  or  serum 
globulin  appears  in  the  urine.  There  are  two  distinct  forms  of  albumin- 
uria, i.  e.,  renal  albuminuria  and  accidental  albuminuria.  Sometimes 
the  terms  "true"  albuminuria  and  "false"  albuminuria  are  substi- 
tuted for  those  just  given.  In  the  renal  type  the  albumin  is  excreted 
by  the  kidneys.  This  is  the  more  serious  form  of  the  malady  and  at 
the  same  time  is  more  frequently  encountered  than  the  accidental 
type.  Among  the  causes  of  renal  albuminuria  are  altered  blood  pres- 
sure in  the  kidneys,  altered  kidney  structure,  or  changes  in  the  com- 
position of  the  blood  entering  the  kidneys,  thus  allowing  the  albumin 
to  diffuse  more  readily.  In  the  accidental  form  of  albuminuria  the 
albumin  is  not  excreted  by  the  kidneys  as  is  the  case  in  the  renal  form 
of  the  disorder,  but  arises  from  the  blood,  lymjjh,  or  some  albumin- 
containing  exudate  coming  into  contact  with  llic  urine  al  some  ])()int 
below  the  kidneys. 

EXPERIMKNTS. 

Heller's  Ring  Test.  Place  5  c.c.  of  cone ciitraicd  UNO,,  in  a 
test-tube,  incline  the  tuijc,  and,  by  means  ol"  a  jiipctlc  allow  llu'  urine 
to  flow  slowly  (hnvn  the  side.  The  li(|uids  should  stratify  with  the 
formation  of  a  u'hite  zone  of  precijjitated  albumin  at  the  point  of 
juncture.      If  the  albumin  is  j)resenl  in  xcry  small  aniouul   the  while 


URINE.  309 

zone  may  not  form  until  the  tube  has  been  allowed  to  stand  for  several 
minutes.  If  the  urine  is  quite  concentrated  a  white  zone,  due  to  uric 
acid  or  urates,  will  form  upon  treatment  with  nitric  acid  as  indicated. 
This  ring  may  be  easily  differentiated  from  the  albumin  ring  by  re- 
peating the  test  after  diluting  the  urine  with  3  or  4  volumes  of  water, 
whereupon  the  ring,  if  due  to  uric  acid  or  urates,  will  not  appear.  It 
is  ordinarily  possible  to  differentiate  between  the  albumin  ring  and 
the  uric  acid  ring  without  diluting  the  urine,  since  the  ring,  when  due 
to  uric  acid,  has  ordinarily  a  less  sharply  defined  upper  border,  is 
generally  broader  than  the  albumin  ring  and  frequently  is  situated  in 
the  urine  above  the  point  of  contact  with  the  nitric  acid.  Concentrated 
urines  also  occasionally  exhibit  the  formation,  at  the  point  of  contact, 
of  a  crystalline  ring  with  very  sharply  defined  borders.  This  is  urea 
nitrate  and  is  easily  distinguished  from  the  "fluffy  "  ring  of  albumin. 
If  there  is  any  difficulty  in  differentiation  a  simple  dilution  of  the  urine 
with  water,  as  above  described,  will  remove  the  difficulty.  Various 
colored  zones,  due  either  to  the  presence  of  indican,  bile  pigments,  or 
to  the  oxidation  of  other  organic  urinary  constituents,  may  form  in 
this  test  under  certain  conditions.  These  colored  rings  should  never 
be  confounded  with  the  white  ring  which  alone  denotes  the  presence 
of  albumin. 

After  the  administration  of  certain  drugs  a  white  precipitate  of 
resin  acids  may  form  at  the  point  of  contact  of  the  two  fluids  and  may 
cause  the  observer  to  draw  wrong  conclusions.  This  ring,  if  composed 
of  resin  acids,  will  dissolve  in  alcohol,  whereas  the  albumin  ring  will 
not  dissolve. 

Weinberger  has  recently  shown  that  a  ring  closely  resembling  the 
albumin  ring  is  often  obtained  in  urines  preserved  by  thymol  when 
subjected  to  Heller's  test.  The  ring  is  due  to  the  formation  of  nitro- 
sothymol  and  possibly  nitrothymol.  If  the  thymol  is  removed  from 
the  urine  by  extraction  with  petroleum  ether^  previous  to  adding 
nitric  acid,  the  ring  does  not  form. 

An  instrument  called  the  alhumoscope  Qwr  is  ma  scope)  has  been 
devised  for  use  in  this  test  and  has  met  with  considerable  favor.  The 
method  of  using  the  albumoscope  is  described  below. 

Use  of  the  Alhumoscope. — This  instrument  is  intended  to  facilitate 
the  making  of  "ring"  tests  such  as  Heller's  and  Roberts'.  In  making 
a  test  about  5  c.c.  of  the  solution  under  examination  is  first  introduced 
into  the  apparatus  through  the  larger  arm  and  the  reagent  used  in  the 

'  Accomplished  readily  by  gently  agitating  equal  volumes  of  petroleum  ether  and  the 
urine  under  examination  for  two  minutes  in  a  test-tube  before  applying  the  test. 


3IO  PHYSIOLOGICAL    CHEMISTRY. 

particular  test  is  then  introduced  through  the  capillary  arm  and  allowed 
to  flow  down  underneath  the  solution  under  examination.  If  a  reason- 
able amount  of  care  is  taken  there  is  no  possibility  of  mixing  the  two 
solutions  and  a  definitely  defined  white  ''ring"  is  easily  obtained  at 
the  zone  of  contact. 

2.  Roberts'  Ring  Test. — Place  5  c.c.  of  Roberts'  reagent^  in  a 
test-tube,  incline  the  tube,  and,  by  means  of  a  pipette,  allow  the 
urine  to  flow  slou'ly  down  the  side.  The  liquids  should  stratify  with 
the  formation  of  a  u'liite  zone  of  precipitated  albumin  at  the  point  of 
juncture.  This  test  is  a  modification  of  Heller's  ring  test  and  is  rather 
more  satisfactory  than  that  test,  since  the  colored  rings  never  form 
and  the  consequent  confusion  is  avoided.  The  allmmoscope  (see 
above)  may  also  be  used  in  making  this  test. 

3.  Spiegler's  Ring  Test. — Place  5  c.c.  of  Spiegler's  reagent^  in 
a  test-tube,  incline  the  tube,  and,  by  means  of  a  pipette,  allow  5  c.c. 
of  urine,  acidified  with  acetic  acid,  to  flow  slowly  down  the  side.  A 
white  zone  will  form  at  the  point  of  contact.  This  is  an  exceedingly 
delicate  test,  in  fact  too  delicate  for  ordinary  clinical  purposes,  since 
it  serves  to  detect  albumin  when  present  in  the  merest  trace  (i  :  250,000) 
and  hence  most  normal  urines  will  give  a  positive  reaction  for  albumin 
when  this  test  is  applied. 

Some  investigators  claim  that  the  delicacy  of  this  test  depends 
upon  the  presence  of  sodium  chloride  in  the  urine,  the  test  losing 
accuracy  if  the  sodium  chloride  content  be  low. 

4.  Jolles'  Reaction. — Shake  5  c.c.  of  urine  with  i  c.c.  of  30  per 
cent  acetic  acid  and  4  c.c.  of  Jolles'  reagent^  in  a  test-tube.  A  white 
precipitate  indicates  the  presence  of  albumin. 

Care  should  be  taken  to  use  the  correct  amount  of  acetic  acid, 
since  the  use  of  too  small  an  amount  may  result  in  the  formation  of 
mercury  combinations  which  may  cause  confusion.  In  the  presence 
of  iodine,  mercuric  iodide  will  form  but  may  readily  be  differentiated 
from  albumin  through  the  fact  that  it  is  soluble  in  alcohol. 

5.  Coagulation  or  Boiling  Test.— (a)  Heat  5  c.c.  of  urine  to 

'  Roberts'  reagent  is  composed  of  i  volume  of  concentrated  UNO.,  and  5  volumes 
of  a  saturated  solution  of  MgSO,. 

^  .Spiegler's  reagent  has  the  following  composition: 

Tartaric  acid 20  grams. 

Mercuric  chloride 40  grams. 

Glycerol 100  grams. 

Distilled  water   1000  grams. 

*  Jolles'  reagent  has  the  following  composition: 

Succinic  acid 40  grams. 

Mercuric  chloride 20  grams. 

Sodium  chloride 20  grams. 

Distilled  water    1000  grams. 


URINE.  311 

boiling  in  a  test-tube.  ,A  precipitate  forming  at  this  point  is  due 
either  to  albumin  or  to  phosphates.  Acidify  the  urine  slightly  by 
the  addition  of  3-5  drops  of  very  dilute  acetic  acid,  adding  the  acid 
drop  by  drop  to  the  hot  solution.  If  the  precipitate  is  due  to  phos- 
phates it  will  disappear  under  these  conditions,  whereas  if  it  is  due 
to  albumin  it  will  not  only  fail  to  disappear  but  wdll  become  more 
flocculent  in  character,  since  the  reaction  of  a  fluid  must  be  acid  to 
secure  the  complete  precipitation  of  the  albumin  by  this  coagulation 
process.  Too  much  acid  should  be  avoided  since  it  will  cause  the 
albumin  to  go  into  solution.  Certain  resin  acids  may  be  precipi- 
tated by  the  acid,  but  the  precipitate  due  to  this  cause  may  be  easily 
differentiated  from  the  albumin  precipitate  by  reason  of  its  solubility 
in  alcohol. 

(6)  A  modification  of  this  test  in  quite  general  use  is  as  follows: 
Fill  a  test-tube  two-thirds  full  of  urine  and  gently  heat  the  upper  half 
of  the  fluid  to  boiling,  being  careful  that  this  fluid  does  not  mix  with 
the  lower  half.  A  turbidity  indicates  albumin  or  phosphates.  Acidify 
the  urine  slightly  by  the  addition  of  3-5  drops  of  dilute  acetic  acid, 
when  the  turbidity,  if  due  to  phosphates,  wdll  disappear. 

Nitric  acid  is  often  used  in  place  of  acetic  acid  in  these  tests.  In 
case  nitric  acid  is  used  ordinarily  1-2  drops  is  sufficient. 

6.  Acetic  Acid  and  Potassium  Ferrocyanide  Test. — To  5  c.c. 
of  urine  in  a  test-tube  add  5-10  drops  of  acetic  acid.  Mix  well  and 
add  potassium  ferrocyanide  drop  by  drop,  until  a  precipitate  forms. 
This  is  a  very  delicate  test.  Schmiedl  claims  that  a  precipitate  of 
Fe(Cn)gK2Zn  or  Fe(Cn)gZn2  is  formed  when  urines  containing  zinc 
are  subjected  to  this  test  and  that  this  precipitate  resembles  the  pre- 
cipitate secured  with  protein  solutions.  In  the  case  of  human  urine  a 
reaction  was  obtained  when  0.000022  gram  of  zinc  per  cubic  centimeter 
was  present.  Schmiedl  further  found  that  the  urine  collected  from 
rabbits  housed  in  zinc-lined  cages  possessed  a  zinc  content  w^hich  was 
sufficient  to  yield  a  ready  response  to  the  test.  Zinc  is  the  only  inter- 
fering substance  so  far  reported. 

7.  Tanret's  Test. — To  5  c.c.  of  urine  in  a  test-tube  add  Tanret's 
reagent^  drop  by  drop  until  a  turbidity  or  precipitate  forms.  This 
is  an  exceedingly  delicate  test.  Sometimes  the  urine  is  stratified 
upon  the  reagent  as  in  Heller's  or  Roberts'  ring  test.  According  to 
Repiton,   urates    interfere   with   the    delicacy   of   this  test.      Tanret, 

^  Tanret's  reagent  is  prepared  as  follows:  Dissolve  1.35  gram  of  mercuric  chloride 
in  25  c.c.  of  water,  add  to  this  solution  3.32  grams  of  potassium  iodide  dissolved  in  25  c.c. 
of  water,  then  make  the  total  solution  up  to  60  c.c.  with  water  and  add  20  c.c.  of  glacial 
acetic  acid  to  the  mixture. 


312  PHYSIOLOGICAL    CHEMISTRY. 

however,  claims  that  urates  do  not  interfete  inasmuch  as  any  pre- 
cipitate due  to  urates  may  be  brought  into  solution  by  heat  whereas 
an  albumin  precipitate  under  the  same  conditions  will  persist.  Tan- 
ret  further  states  that  mucin  interferes  with  the  delicacy  of  the  test  and 
that  it  should  therefore  be  removed  from  the  urine  under  examination 
by  acidification  with  acetic  acid  and  filtration  before  testing  for 
albumin. 

8.  Sodium  Chloride  and  Acetic  Acid  Test. — Mix  two  volumes 
of  urine  and  one  \olume  of  a  saturated  solution  of  sodium  chloride 
in  a  test-tube,  acidify  with  acetic  acid,  and  heat  to  boiling.  The  pro- 
duction of  a  cloudiness  or  the  formation  of  a  precipitate  indicates 
the  presence  of  albumin.  The  resin  acids  may  interfere  here  as  in 
the  ordinary  coagulation  test  (page  310),  but  they  may  be  easily 
differentiated  from  albumin  by  means  of  their  solubility  in  alcohol. 

9.  Potassium  Iodide  Test/ — Dilute  5  c.c.  of  the  urine  under 
examination  with  to  c.c.  of  water  and  stratify  this  mixture  upon  a 
potassium  iodide  solution  made  slightly  acid  with  acetic  acid.  In 
the  presence  of  0.01-0.02  per  cent  of  albumin  a  white  ring  forms 
immediately.  If  the  test  be  allowed  to  stand  two  minutes  after  the 
stratification  it  will  serve  to  detect  0.005  per  cent  of  albumin. 

GLOBULIN. 

Serum  globulin  is  not  a  constituent  of  normal  urine  l)ul  frecjuently 
occurs  in  the  urine  under  pathological  conditions  and  is  ordinarily 
associated  with  serum  albumin.  In  albuminuria  globulin  in  varying 
amounts  often  accompanies  the  albumin,  and  the  clinical  significance 
of  the  two  is  very  similar.  Under  certain  conditions  globulin  may 
occur  in  the  urine  unaccompanied  by  albumin. 

Experiments. 

Globulin  will  respond  to  all  the  tests  just  outlined  under  Albumin. 
If  it  is  desirable  to  dilTerentiate  between  albumin  and  globulin  in 
any  urine  the  following  processes  may  be  employed: 

I.  Saturation  with  Magnesium  Sulphate.  Place  25  c.c.  of 
neutral  urine  in  a  small  beaker  and  add  ])ul\eri/ed  magnesium  sul- 
f>hale  in  suhstante  to  the  j)oinl  of  saturation.  If  the  ])r()tein  ])resent 
is  globulin  it  will  prct  ipiiaic  at  this  point.  If  no  precipitate  is  pro- 
duced acidify  the  saturated  solution  with  acetic  a(  id  and  warm  gently. 
Albumin  will  he  y)re(ipitated  if  yjresent. 

'  I'lianii.  Zlf^.,  54,  1^.  (>ij. 


URINE.  313 

The  above  procedure  may  be  used  to  separate  globulin  and  albu- 
min if  present  in  the  same  urine.  To  do  this  filter  off  the  globulin 
after  it  has  been  precipitated  by  the  magnesium  sulphate,  then  acidify 
the  clear  solution  and  warm  gently  as  directed.  Note  the  formation 
of  the  albumin  precipitate. 

2.  Half-saturation  with  Ammonium  Sulphate. — Place  25  c.c. 
of  neutral  urine  in  a  small  beaker  and  add  an  equal  volume  of  a 
saturated  solution  of  ammonium  sulphate.  Globulin,  if  present, 
will  be  precipitated.  If  no  precipitate  forms  add  ammonium  sul- 
phate in  substance  to  the  point  of  saturation.  If  albumin  is  present 
it  will  be  precipitated  upon  saturation  of  the  solution  as  just  indicated. 
This  method  may  also  be  used  to  separate  globulin  and  albumin  when 
they  occur  in  the  same  urine. 

Frequently  in  urine  which  contains  a  large  amount  of  urates  a 
precipitate  of  ammonium  urate  may  occur  when  the  ammonium  sul- 
phate solution  is  added  to  the  urine.  This  urate  precipitate  should 
not  be  confounded  with  the  precipitate  due  to  globulin.  The  two 
precipitates  may  be  differentiated  by  means  of  the  fact  that  the  urate 
precipitate  ordinarily  appears  only  after  the  lapse  of  several  minutes 
whereas  the  globulin  generally  precipitates  at  once. 

PROTEOSE  AND  PEPTONE. 

Proteoses,  particularly  deutero-proteose  and  hetero-proteose,  have 
frequently  been  found  in  the  urine  under  various  pathological  con- 
ditions such  as  diphtheria,  pneumonia,  intestinal  ulcer,  carcinoma, 
dermatitis,  osteomalacia,  atrophy  of  the  kidneys,  and  in  sarcomata 
of  the  bones  of  the  trunk.  "  Bence- Jones'  protein,"  a  proteose-like 
substance,  is  of  interest  in  this  connection  and  its  appearance  in  the 
urine  is  believed  to  be  of  great  diagnostic  importance  in  cases  of  multi- 
ple myeloma  or  myelogenic  osteosarcoma.  By  some  investigators 
this  protein  is  held  to  be  a  variety  of  hetero-proteose  whereas  others 
claim  that  it  possesses  albumin  characteristics. 

Peptone  -certainly  occurs  much  less  frequently  as  a  constituent  of 
the  urine  than  does  proteose,  in  fact  most  investigators  seriously  ques- 
tion its  presence  under  any  conditions.  There  are  many  instances 
of  peptonuria  cited  in  the  early  literature,  but  because  of  the  uncer- 
tainty in  the  conception  of  what  really  constituted  a  peptone  it  is  prob- 
able that  in  many  cases  of  so-called  peptonuria  the  protein  present 
was  really  proteose. 


314  physiological  chemistry. 

Experiments. 

1.  Boiling  Test. — Make  the  ordinary  coagulation  test  according 
to  the  directions  given  under  Albumin,  page  310.  If  no  coagulable 
protein  is  found  allow  the  boiled  urine  to  stand  and  note  the  gradual 
appearance,  in  the  cooled  fluid,  of  a  flaky  precipitate  of  proteose.  This 
is  a  crude  test  and  should  never  be  relied  upon. 

2.  Schulte's  Method. — Acidify  50  c.c.  of  urine  with  dilute  acetic 
acid  and  filter  oft'  any  precipitate  of  nucleoprotein  which  may  form. 
Xow  test  a  few  cubic  centimeters  of  the  urine  for  coagulable  pro- 
tein, by  tests  2  and  5  under  Albumin,  p.  310.  If  coagulable  protein 
is  present  remove  it  by  coagulation  and  filtration  before  proceed- 
ing. Introduce  25  c.c.  of  the  urine,  freed  from  coagulable  pro- 
tein, into  150  c.c.  of  absolute  alcohol  and  allow  it  to  stand  for  12- 
24  hours.  Decant  the  supernatant  fluid  and  dissolve  the  precipitate 
in  a  small  amount  of  hot  water.  Now  filter  this  solution,  and  after 
testing  again  for  nucleoprotein  with  very  dilute  acetic  acid,  try  the  biuret 
test.     If  this  test  is  positive  the  presence  of  proteose  is  indicated.^ 

Urobilin  does  not  ordinarily  interfere  with  this  test  since  it  is  al- 
most entirely  dissolved  by  the  absolute  alcohol  when  the  proteose  is 
precipitated. 

3.  V.  Aider's  Method. — Acidify  10  c.c.  of  urine  with  hydro- 
chloric acid,  add  phosphotungstic  acid  until  no  more  precipitate 
forms  and  centrifugate^  the  solution.  Decant  the  supernatant  fluid, 
add  some  absolute  alcohol  to  the  precipitate,  and  centrifugate  again. 
This  washing  with  alcohol  is  intended  to  remove  the  urobilin  and 
hence  should  be  continued  so  long  as  the  alcohol  exhibits  any  colora- 
tion whatever.  Now  suspend  the  precipitate  in  water  and  add  potas- 
sium hydroxide  to  bring  it  into  solution.  At  this  point  the  solution  may 
be  blue  in  color,  in  which  case  decolorization  may  be  secured  by  gently 
heating.  Apply  the  biuret  test  to  the  cool  solution.  A  positive  biuret 
test  indicates  the  presence  of  proteoses. 

4.  Detection  of  "Bence-Jones'  Protein." — Heat  the  suspected 
urine  very  gently,  carefully  noting  the  tem])eraturc.  At  as  low  a 
temperature  as  40'^  C.  a  turbirlily  may  be  obser\e(l,  and  as  the  tem- 
perature is  raised  to  about  60"^  C.  a  flocculent  precipitate  forms  and 
clings  to  the  sides  of  the  test-tube.  If  the  urine  is  now  acidified  very 
slightly  with  acetic  acid  and  the  temy)erature  further  raised  to  100°  C. 

'  If  it  is  consiflcrer]  desirable  to  test  for  |;L]Uonc  the  |)rotcost;  may  Ijc  removed  hy 
saturation  with  (Nri,)._,SO,  according  to  the  directions  ^iv(;n  on  paj^c  112  and  tlic  iiltrate 
tested  for  peptone  by  the  Ijiuret  test. 

*  If  not  convenient  to  use  a  centrifuge  the  |)re(  ipitate  may  be  fillerc-d  off  and  washed 
on  the  filter  paper  with  alcohol. 


URINE.  315 

the  precipitate  at  least  partly  disappears;  it  will  return  upon  cooling 
the  tube. 

This  property  of  precipitating  at  so  low  a  temperature  and  of 
dissolving  at  a  higher  temperature  is  typical  of  "Bence- Jones'  pro- 
tein" and  may  be  used  to  differentiate  it  from  all  other  forms  of  protein 
material  occurring  in  the  urine. 

NUCLEOPROTEIN. 

There  has  been  considerable  controversy  as  to  the  proper  classi- 
fication for  the  protein  body  which  forms  the  "nubecula"  of  normal 
urine.  By  different  investigators  it  has  been  called  mucin,  mucoid^ 
phosphoprotein,  niicleo albumin,  and  nucleoprotein.  Of  course,  accord- 
ing to  the  modern  acceptation  of  the  meanings  of  these  terms  they 
cannot  be  synonymous.  Mucin  and  mucoid  are  glycoproteins  and 
hence  contain  no  phosphorus  (see  p.  85),  whereas  phosphoproteins 
and  nucleoproteins  are  phosphorized  bodies.  It  may  possibly  be 
that  both  these  forms  of  protein,  i.  e.,  the  glycoprotein  and  the  phos- 
phorized type,  occur  in  the  urine  under  certain  conditions  (see  page 
284).  In  this  connection  we  will  use  the  term  nucleoprotein.  The 
pathological  conditions  under  which  the  content  of  nucleoprotein  is 
increased  includes  all  affections  of  the  urinary  passages  and  in  particular 
pyelitis,  nephritis,  and  inflammation  of  the  bladder. 

Experiments. 

1.  Detection  of  Nucleoprotein. — Place  10  c.c.  of  urine  in  a 
small  beaker,  dilute  it  with  three  volumes  of  water  to  prevent  pre- 
cipitation of  urates,  and  make  the  reaction  very  strongly  acid  with 
acetic  acid.  If  the  urine  becomes  turbid  it  is  an  indication  that  nucleo- 
protein is  present. 

If  the  urine  under  examination  contains  albumin  the  greater  por- 
tion of  this  substance  should  be  removed  by  boiling  the  urine  before 
testing  it  for  the  presence  of  nucleoprotein. 

2.  Ott's  Precipitation  Test. — Mix  25  c.c.  of  the  urine  with  an 
equal  volume  of  a  saturated  solution  of  sodium  chloride  and  slowly 
add  Almen's  reagent.^  In  the  presence  of  nucleoprotein  a  voluminous 
precipitate  forms. 

BLOOD. 

The  pathological  conditions  in  which  blood  occurs  in  the  urine 
may  be  classified  under  the  two  divisions  hcematuria  and  hamoglohin- 

^  Dissolve  5  grams  of  tannin  in  240  c.c.  of  50  per  cent  alcohol  and  add  10  c.c.  of  25  per 
cent  acetic  acid. 


3l6  PHYSIOLOGICAL    CHEMISTRY. 

un'a.  In  htematuria  we  are  able  to  detect  not  only  the  haemoglobin 
but  the  unruptured  corpuscles  as  well,  whereas  in  hemoglobinuria 
the  pigment  alone  is  present.  Hasmaturia  is  brought  about  through 
blood  passing  into  the  urine  because  of  some  lesion  of  the  kidney  or 
of  the  urinary  tract  below  the  kidney.  Hoemoglobinuria  is  brought 
about  through  haemolysis,  /.  e.,  the  rupturing  of  the  stroma  of  the 
erythrocyte  and  the  liberation  of  the  haemoglobin.  This  may  occur  in 
scurvy,  typhus,  pyemia,  purpura,  and  in  other  diseases.  It  may  also 
occur  as  the  result  of  a  burn  covering  a  considerable  area  of  the  body, 
or  may  be  brought  about  through  the  action  of  certain  poisons  or  by 
the  injections  of  various  substances  having  the  power  of  dissolving  the 
ervthroctyes.     Transfusion  of  blood  may  also  cause  haemoglobinuria. 

Experiments. 

1.  Heller's  Test, — Render  lo  c.c.  of  urine  strongly  alkaline  with 
potassium  hydroxide  solution  and  heat  to  boiling.  Upon  allowing 
the  heated  urine  to  stand  a  precipitate  of  phosphates,  colored  red 
by  the  contained  haematin,  is  formed.  It  is  ordinarily  well  to  make  a 
''control"  experiment  using  normal  urine,  before  coming  to  a  final 
decision. 

Certain  substances,  such  as  cascara  sagrada,  rhubarb,  santonin, 
and  senna,  cause  the  urine  to  give  a  similar  reaction.  Reactions  due 
to  such  substances  may  be  differentiated  from  the  true  ])lood  reac- 
tion by  the  fact  that  both  the  precipitate  and  the  pigment  of  the 
former  reaction  disappear  when  treated  with  acetic  acid,  whereas  if 
the  color  is  due  to  haematin  the  acid  will  only  dissolve  the  precipitate 
of  jjhosphates  and  leave  the  pigment  undissoKed. 

2.  Teichmann's  Haemin  Test.  —Place  a  small  drop  of  the  sus- 
pected urine  or  a  small  amount  of  the  moist  sediment  on  a  micro- 
scopic slide,  add  a  minute  grain  of  sodium  •  chloride  and  carefully 
e\'aporate  to  dryness  over  a  low  flame.  Put  a  coverglass  in  place, 
run  underneath  it  a  drop  of  glacial  acetic  acid,  and  warm  gently  until 
the  formation  of  gas  bubbles  is  observed.  Cool  the  pre])aration,  ex- 
amine under  the  microscope,  and  conip.irc  llic  form  of  the  crystals 
with  thfjse  reprorluced  in  Figs.  58  and  st;,  page  i(;.|.  (See  Atkinson 
and  Kenflall's  modifkatifjn,  p.  193.) 

3.  Heller-Teichmann  Reaction.  I'lodiuc  the  ])igmented  i)rc- 
cipitate  according  to  directions  gi\en  in  Heller's  test  above.  If 
there  is  a  copious  precij;itate  of  jjhosjjhates  and  Init  little  ])igment 
the  phosphates  may  be  dissolved  by  treatment  with  acetic  acid  and 


URINE.  317 

the  residue  used  in  the  formation  of  the  hsemin  crystals  according  to 
directions  in  Experiment  2,  p.  316. 

4.  V.  Zeynek  and  Nencki's  Haemin  Test. — To  10  c.c.  of  the 
urine  under  examination  add  acetone  until  no  more  precipitate  forms. 
Filter  off  the  precipitate  and  extract  it  with  10  c.c.  of  acetone  rendered 
acid  with  2-3  drops  of  hydrochloric  acid.  Place  a  drop  of  the  resulting 
colored  extract  on  a  slide,  immediately  place  a  coverglass  in  position, 
and  examine  under  the  microscope.  Compare  the  form  of  the  crystals 
with  those  shown  in  Figs.  58  and  59,  page  194.  Haemin  crystals 
produced  by  this  manipulation  are  sometimes  very  minute,  thus  render- 
ing it  difhcult  to  determine  the  exact  form  of  the  crystal. 

5.  Schalfijew's  Haemin  Test. — Place  20  c.c.  of  glacial  acetic 
acid  in  a  small  beaker  and  heat  to  80°  C.  Add  5  c.c.  of  the  urine  under 
examination,  raise  the  temperature  to  80  °  C,  and  stand  the  mixture 
aside  to  cool.  Examine  the  crystals  under  the  microscope  and  com- 
pare them  with  those  shown  in  Figs.  58  and  59,  page  194. 

6.  Guaiac  Test. — Place  5  c.c.  of  urine  in  a  test-tube  and  by  means 
of  a  pipette  introduce  a  freshly  prepared  alcoholic  solution  of  guaiac 
(strength  about  1:60)  into  the  fluid  until  a  turbidity  results,  then  add 
old  turpentine  or  hydrogen  peroxide,  drop  by  drop,  until  a  blue  color 
is  obtained.  This  is  a  very  delicate  test  w^hen  properly  performed. 
Buckmaster  has  recently  suggested  the  use  of  guaiaconic  acid  instead 
of  the  solution  of  guaiac.  See  discussion  on  page  188  and  test  on 
page  191. 

7.  Schtimni's  Modification  of  the  Guaiac  Test. — To  about  5 
c.c.  of  urine^  in  a  test-tube  add  about  10  drops  of  a  freshly  prepared 
alcoholic  solution  of  guaiac.  Agitate  the  tube  gently,  add  about  20 
drops  of  old  turpentine,  subject  the  tube  to  a  thorough  shaking,  and 
permit  it  to  stand  for  about  2-3  minutes.  A  blue  color  indicates  the 
presence  of  blood  in  the  solution  under  examination.  In  case  there 
is  not  sufficient  blood  to  yield  a  blue  color  under  these  conditions,  a  few 
c.c.  of  alcohol  should  be  added  and  the  tube  gently  shaken,  whereupon 
a  blue  coloration  will  appear  in  the  upper  alcohol-turpentine  layer. 

A  control  test  should  always  be  made  using  water  in  place  of  urine. 
In  the  detection  of  very  minute  traces  of  blood  only  3—5  drops  of  the 
guaiac  solution  should  be  employed. 

8.  Adler's  Benzidine  Reaction. — This  is  one  of  the  most  delicate 
of  the  reactions  for  the  detection  of  blood.  Different  benzidine  prep- 
arations vary  greatly  in  their  sensitiveness,  however.     Inasmuch  as 

^  Alkaline  urine  should  be  made  slightly  acid  with  acetic  acid  as  the  blue  end-reaction 
is  very  sensitive  to  alkah. 


3l8  PHYSIOLOGICAL   CHEMISTRY. 

benzidine  solutions  change  readily  upon  contact  with  light,  it  is 
essential  that  they  be  kept  in  a  dark  place.  The  test  is  performed  as 
follows :  To  a  saturated  solution  of  benzidine  in  alcohol  or  glacial 
acetic  acid  add  an  equal  volume  of  3  per  cent  hydrogen  peroxide  and 
I  c.c.  of  the  urine  under  examination.  If  the  mixture  is  not  already 
acid,  render  it  so  with  acetic  acid,  and  note  the  appearance  of  a  green 
or  blue  color.  A  control  test  should  be  made  substituting  water  for 
the  urine. 

Often  when  urines  containing  a  small  amount  of  blood  are  tested 
by  this  reaction,  the  mixture  is  rendered  so  turbid  as  to  make  it  difficult 
to  decide  as  to  the  presence  of  a  faint  green  color.  Such  urines  should 
be  extracted  with  an  ether-acetic  acid  solution  and  the  resulting 
extract  washed  with  water  before  the  test  is  applied  to  it.  The  sensi- 
tiveness of  the  benzidine  reaction  is  greater  when  applied  to  aqueous 
solutions  than  when  applied  to  the  urine. 

9.  Spectroscopic  Examination. — Submit  the  urine  to  a  spectro- 
scopic examination  according  to  the  directions  given  on  page  198, 
looking  especially  for  the  absorption-bands  of  oxyhaemoglobin  and 
methaemoglobin  (see  Absorption  Spectra,  Plate  I.). 

BILE. 

Both  the  pigments  and  the  acids  of  the  bile  may  be  detected  in  the 
urine  under  certain  pathological  conditions.  Of  the  pigments,  bilirubin 
is  the  only  one  which  has  been  p()siti\'ely  identified  in  fresh  urine;  the 
other  pigments,  when  present,  are  probably  derived  from  the  bilirubin. 
A  urine  containing  bile  may  be  yellowish-green  to  brown  in  color  and 
when  shaken  foams  readily.  The  staining  of  the  various  tissues  of  the 
body  through  the  absorption  of  bile  due  to  occlusion  of  the  bile  duct 
causes  a  condition  known  as  icterus  or  jaundice.  Bile  is  always 
present  in  the  urine  under  such  conditions  unless  the  amount  of  bile 
reaching  the  tissues  is  extremely  small. 

Experiments. 

Tests  for  Bile  Pigments. 

J.  Gmelin's  Test. — To  about  5  c.c.  of  concentrated  nitric  acid  in 
a  test-tube  add  an  equal  volume  of  urine  carefully  so  that  the  two 
fluids  flo  not  mix.  At  the  ]joint  of  contact  nolo  the  \;irious  colored 
rings,  f^reen,  blue,  violet,  red,  unci  reddish-yelUnv. 

2.  Rosenbach's  Modification  of  Gmelin's  Test. — Filter  5  c.c. 
of  urine  through  a  small  filter  paper,    introduce  a  drop  of  concentrated 


URINE.  319 

nitric  acid  into  the  cone  of  the  paper  and  observe  the  succession  of 
colors  as  given  in  Gmelin's  test. 

3.  Nakayama's  Reaction. — To  5  c.c.  of  urine  in  a  test-tube  add 
an  equal  volume  of  a  10  per  cent  solution  of  barium  chloride.  Cen- 
trifugate  the  mixture,  pour  off  the  supernatant  fluid,  and  heat  the 
precipitate  with  2  c.c.  of  Nakayama's  reagent.^  In  the  presence  of 
bile  pigments  the  solution  assumes  a  blue  or  green  color. 

3.  Huppert's  Reaction. — Thoroughly  shake  equal  volumes  of 
urine  and  milk  of  lime  in  a  test-tube.  The  pigments  unite  with  the 
calcium  and  are  precipitated.  Filter  off  the  precipitate,  wash  it  with 
water,  and  transfer  to  a  small  beaker.  Add  alcohol  acidified  slightly 
with  hydrochloric  acid  and  warm  upon  a  water-bath  until  the  solution 
becomes  colored  an  emerald  green. 

According  to  Steensma,  this  procedure  may  give  negative  results' 
even  in  the  presence  of  the  pigments,  owing  to  the  fact  that  the  acid- 
alcohol  is  not  a  sufficiently  strong  oxidizing  agent.  He  therefore 
suggests  the  addition  of  a  drop  of  a  0.5  per  cent  solution  of  sodium 
nitrite  to  the  acid-alcohol  mixture  before  warming  on  the  water-bath. 
Try  this  modification  also. 

4.  Salkowski's  Test. — Render  5  c.c.  of  urine  alkaline  with  a  few 
drops  of  a  10  per  cent  sodium  carbonate  solution  and  add  a  10  per 
cent  solution  of  calcium  chloride,  drop  by  drop,  until  the  supernatant 
fluid  exhibits  the  normal  urinary  color  when  the  contents  of  the  test-tube 
are  thoroughly  mixed.  Filter  off  the  precipitate,  and  after  washing  it 
place  it  in  a  second  tube  with  95  per  cent  alcohol.  Acidify  the  alcohol 
with  hydrochloric  acid  and,  if  necessary,  shake  the  tube  to  bring  the 
precipitate  into  solution.  Heat  the  solution  to  boiling  and  observe  the 
appearance  of  a  green  color  which  changes  through  blue  and  violet  to 
red;  if  no  bile  is  present  the  solution  does  not  undergo  any  color 
change.  This  test  will  frequently  exhibit  greater  delicacy  than  Gmelin's 
test.  Steensma's  suggestions  mentioned  under  Huppert's  Reaction, 
above,  apply  in  connection  with  this  test  also. 

5.  Hammarsten's  Reaction. — To  about  5  c.c.  of  Hammarsten's 
reagent^  in  a  small  evaporating  dish  add  a  few  drops  of  urine.  A 
green  color  is  produced.  If  more  of  the  reagent  is  now  added  the  play 
of  colors  as  noted  in  Gmelin's  test  may  be  obtained. 

6.  Smith's  Test. — To  2-3  c.c.  of  urine  in  a  test-tube  add  carefully 

^  Prepared  by  combining  gg  c.c.  of  alcohol  and  i  c.c.  of  fuming  hydrochloric  acid 
containing  4  grams  of  ferric  chloride  per  hter. 

-  Hammarsten's  reagent  is  made  by  mixing  i  volume  of  25  per  cent  nitric  acid  and  19 
volumes  of  25  per  cent,  hydrochloric  acid  and  then  adding  i  volume  of  this  acid  mixture 
to  4  volumes  of  95  per  cent  alcohol. 


320  PHYSIOLOGICAL    CHEMISTRY. 

about  5  c.c.  of  dilute  tincture  of  iodine  (i :  lo)  so  that  the  fluids  do  not 
mix.    A  green  ring  is  observed  at  the  point  of  contact. 

7.  Salkowski-Schippers  Reaction. — Xculralize  the  acidity  of 
10  c.c.  of  the  urine  under  examination  with  a  few  drops  of  a  dilute 
solution  of  sodium  carbonate,  and  add  5  drops  of  a  20  per  cent  solution 
of  sodium  carbonate  and  10  drops  of  a  20  per  cent  solution  of  calcium 
chloride.  Filter  off  the  resultant  precipitate  upon  a  hardened  filter 
paper  and  wash  it  with  water.  Remove  the  precipitate  to  a  small 
porcelain  dish,  add  3  c.c.  of  an  acid-alcohol  mixture^  and  a  few  drops 
of  a  dilute  solution  of  sodium  nitrite  and  heat.  The  production  of  a 
green  color  indicates  the  presence  of  bile  pigments. 

8.  Bonanno's  Reaction.' — Place  5-10  c.c.  of  the  urine  under 
examination  in  a  small  porcelain  evaporating  dish  and  add  a  few 
drops  of  Bonanno's  reagent.^  If  bile  is  present  an  emerald-green 
color  will  develop.  Bonanno  says  the  reaction  is  not  interfered  with 
by  any  known  normal  or  pathological  urinary  constituent. 

Tests  for  Bile  Acids. 

1.  Pettenkofer's  Test. — To  5  c.c.  of  urine  in  a  test-tube  add  5 
drops  of  a  5  per  cent  solution  of  sucrose.  Now  incline  the  tube,  run 
about  2-3  c.c.  of  concentrated  sulphuric  acid  carefully  down  the  side 
and  note  the  red  ring  at  the  point  of  contact.  Upon  slightly  agitating 
the  contents  of  the  tube  the  whole  solution  gradually  assumes  a  reddish 
color.  As  the  tube  becomes  warm,  it  should  be  c(Joled  in  running 
water  in  order  that  the  temperature  may  not  rise  al)()ve  70°  C. 

2.  Mylius's  Modification  of  Pettenkofer's  Test. — To  approx- 
imately 5  c.c.  of  urine  in  a  test-tube  add  3  drops  of  a  very  dilute  (i :  1000) 
aqueous  solution  of  furfurol, 

HC CH 

II  II 

HC         CCHO. 


O 

Now  incline  the  tube,  run  about  2  3  ex.  of  {'oncentraled  sulphuric 
acid  carefully  down  the  side  and  note  the  red  ring  as  abo\e.  In  this 
case  also,  upon  shaking  the  tube,  the  whole  solution  is  colored  red. 
Keep  the  temperature  below  70°  C  as  before. 

'  Made  \>y  adding  5  c.c.  of  contcnlralcd  hydroclilorii  arid  lo  ij^  (  .(  .  nf  ()(>  \n-v  ( cnl 
alcohol. 

*  II  Tommasi,  2,  No.  21. 

'This  reagent  may  be  [jrejjared  hy  dissolving  2  grams  of  sodium  niuitc  in  100  c.c. 
of  concentrated  hydrochloric  add. 


URINE.  321 

3.  Neukomm's  Modification  of  Pettenkofer's  Test. — To  a  few 
drops  of  urine  in  an  evaporating  dish  add  a  trace  of  a  dilute  sucrose 
solution  and  one  or  more  drops  of  dilute  sulphuric  acid.  E\-aporate 
on  a  water-bath  and  observe  the  development  of  a  violet  color  at  the 
edge  of  the  evaporating  mixture.  Discontinue  the  evaporation  as 
soon  as  the  color  is  observed. 

4.  V.  Udransky's  Test. — To  5  c.c.  of  urine  in  a  test-tube  add 
3-4  drops  of  a  very  dilute  (i  :  1000)  aqueous  solution  of  furfurol. 
Place  the  thumb  over  the  top  of  the  tube  and  shake  until  a  thick  foam 
is  formed.  By  means  of  a  small  pipette  add  2-3  drops  of  concentrated 
sulphuric  acid  to  the  foam  and  observe  the  dark  pink  coloration 
produced. 

6.  Hay's  Test. — This  test  is  based  upon  the  principle  that  bile 
acids  have  the  property  of  reducing  the  surface  tension  of  fluids  in 
which  they  are  contained.  The  test  is  performed  as  follows:  Cool 
about  10  c.c.  of  urine  in  a  test-tube  to  17°  C.  or  lower,  and  sprinkle  a 
little  finely  pulverized  sulphur  upon  the  surface  of  the  fluid.  The 
presence  of  bile  acids  is  indicated  if  the  sulphur  sinks  to  the  bottom 
of  the  liquid,  the  rapidity  with  which  the  sulphur  sinks  depending 
upon  the  amount  of  bile  acids  present  in  the  urine.  The  test  is  said 
to  react  with  bile  acids  when  the  latter  are  present  in  the  proportion 
I  :  120,000. 

Some  investigators  claim  that  it  is  impossible  to  differentiate 
between  bile  acids  and  bile  pigments  by  this  test. 

CH3 

ACETONE,    C  =  0. 

CH3 

It  was  formerly  very  generally  believed  that  acetone  appeared  in 
the  urine  under  pathological  conditions  because  of  increased  protein 
decomposition.  It  is  now  generally  thought  that,  in  man,  the  output 
of  acetone  arises  principally  from  the  breaking  down  of  fatty  tissues 
or  fatty  foods  within  the  organism.  The  quantity  of  acetone  elimi- 
nated has  been  shown  to  increase  when  the  subject  is  fed  an  abundance 
of  fat-containing  food  as  well  as  during  fasting,  whereas  a  replace- 
ment of  the  fat  with  carbohydrates  is  followed  by  a  marked  decrease 
in  the  acetone  excretion.  Conditions  are  different  with  certain  of  the 
lower  animals.  With  the  dog,  for  instance,  the  output  of  acetone  is 
not  diminished  when  the  animal  is  fed  upon  a  carbohydrate  diet,  is 


322  PHYSIOLOGICAL   CHEMISTRY. 

decreased  during  fasting,  and  increased  when  the  animal  is  fed  upon 
a  diet  of  meat. 

Acetone  and  the  closely  related  bodies,  ,5-oxybutyric  acid  and 
diacetic  acid,  are  generally  classified  as  the  acetone  bodies.  They  are 
all  associated  with  a  deranged  metabolic  function  and  may  appear 
in  the  urine  together  or  separately,  depending  upon  the  conditions. 
Acetone  and  diacetic  acid  may  occur  alone  in  the  urine  but  /3-oxy- 
butyric  acid  is  never  found  except  in  conjunction  with  one  or  the 
other  of  these  bodies.  Acetone  and  diacetic  acid  arise  chiefly  from 
the  oxidation  of  /3-oxybutyric  acid.  The  relation  existing  between 
these  three  bodies  is  shown  in  the  following  reactions: 

(a)   CH3. CH(OH). CH^. COOH+  O  =  CH3CO.  CH^.  COOH  +  H^O. 

p-oxybutyric  acid.  Diacetic  acid. 

{h)  CH3CO.CH3.COOH=(CH3)2CO  +  C02. 

Diacetic  acid.  Acetone. 

Acetone,  chemically  considered,  is  a  ketone,  di-methyl  ketone. 
When  pure  it  is  a  liquid  which  possesses  a  characteristic  aromatic 
fruit-like  odor,  boils  at  56-57°  C.  and  is  miscible  with  water,  alcohol, 
or  ether  in  all  proportions.  Acetone  is  a  physiological  as  well  as  a 
pathological  constituent  of  the  urine  and  under  normal  conditions 
the  daily  output  is  about  0.01-0.03  gram. 

Pathologically,  the  elimination  of  acetone  is  often  greatly  in- 
creased and  at  such  times  a  condition  of  acetonuria  is  said  to  exist. 
This  pathological  acetonuria  may  accompany  diabetes  mellitus, 
scarlet  fever,  typhoid  fever,  pneumonia,  nephritis,  phosphorus  poison- 
ing, grave  anaemias,  fasting,  and  a  deranged  digestive  function;  it  also 
frequently  accompanies  auto-intoxication  and  chloroform  and  ether 
anaesthesia.  The  types  of  acetonuria  most  frcquentlv  met  with  are 
those  noted  in  febrile  conditions  and  in  advanced  cases  of  diabetes 
mellitus. 

Experiments. 

I.  Isolation  from  the  Urine.  —In  order  to  facilitate  the  detection 
of  acetone  in  the  urine,  the  sj^ccimen  under  examination  should  be 
distilled  and  the  tests  as  given  below  a])j)lied  to  the  resulting  distillate. 
If  it  is  not  convenient  to  distil  the  urine,  the  tests  may  be  conducted 
upon  the  undistilled  fluid.  To  obtain  an  acetone  distillate  proceed 
as  follows:  Place  100-250  c.c.  of  urine  in  a  distillation  flask  or  retort 
and  render  it  acid  with  acetic  acid.    Collccl  about  one  third  of  the  orig- 


URINE. 


Z^Z 


inal  volume  of  fluid  as  a  distillate,  add  5  drops  of  10  per  cent  hydro- 
chloric acid  and  redistil  about  one-half  of  this  volume.  With  this 
final  distillate  conduct  the  tests  as  given  below. 

2.  Gunning's  Iodoform  Test. — To  about  5  c.c.  of  the  urine  or 
distillate  in  a  test-tube  add  a  few  drops  of  Lugol's  solution^  or  ordi- 
nary iodine  solution  (I  in  KI)  and  enough  NH^OH  to  form  a  black 
precipitate  (nitrogen  iodide).  Allow  the  tube  to  stand  (the  length  of 
time  depending  upon  the  content  of  acetone  in  the  fluid  under  examina- 
tion) and  note  the  formation  of  a  yellowish  sediment  consisting  of 
iodoform.  Examine  the  sediment  under  the  microscope  and  compare 
the  form  of  the  crystals  with  those  shown  in  Fig.  6,  p.  42.  If  the  crystals 
are  not  well  formed  recrystallize  them  from  ether  and  examine  again. 
The  crystals  of  iodoform  should  not  be  confounded  with  those  of 
stellar  phosphate  (Fig.  76,  p.  220)  which  may  be  formed  in  this  test, 
particularly  if  made  upon  the  undis tilled  urine.  This  test  is  preferable 
to  Lieben  's  test  (4)  since  no  substance  other  than  acetone  will  produce 
iodoform  when  treated  according  to  the  directions  for  this  test;  both 
alcohol  and  aldehyde  yield  iodoform  when  tested  by  Lieben's  test. 

Gunning's  test  is  rather  the  most  satisfactory  test  yet  suggested 
for  the  detection  of  acetone,  and  may  be  used  with  good  results  even 
upon  the  undistilled  urine.  In  some  instances  where  the  amount  of 
acetone  present  is  very  small  it  is  necessary  to  allow  the  tube  to  stand 
24  hours  before  making  the  examination  for  iodoform  crystals.  This 
test  serves  to  detect  acetone  when  present  in  the  ratio  i  :  100,000. 

3.  Legal's  Test. — Introduce  about  5  c.c.  of  the  urine  or  distillate 
into  a  test-tube,  add  a  few  drops  of  a  freshly  prepared  aqueous  solution 
of  sodium  nitroprusside  and  render  the  mixture  alkaline  with  potas- 
sium hydroxide.  A  ruby  red  color,  due  to  creatinine,  a  normal  urinary 
constituent,  is  produced  (see  Weyl's  test,  p.  273).  Add  an  excess  of 
acetic  acid  and  if  acetone  is  present  the  red  color  will  be  intensified, 
whereas  in  the  absence  of  acetone  a  yellow  color  will  result.  Make 
a  control  test  upon  normal  urine  to  show  that  this  is  so.  A  similar  red 
color  may  be  produced  by  paracresol  in  urines  containing  no  acetone. 

4.  Lieben's  Test. — Introduce  5  c.c.  of  the  urine  or  distillate  into 
a  test-tube,  render  it  alkaline  with  potassium  hydroxide  and  add  1-2 
c.c.  of  iodine  solution  drop  by  drop.  If  acetone  is  present  a  yellowish 
precipitate  of  iodoform  will  be  produced.  Identify  the  iodoform  by 
means  of  its  characteristic  odor  and  its  typical  crystalline  form  (see 
Fig.  6,  p.  42).     While  fully  as  delicate  as  Gunning's  test  (2)  this  test 

'  Lugol's  solution  may  be  prepared  by  dissolving  4  grams  of  iodine  and  6  grams  of 
potassium  iodide  in  100  c.c.  of  distilled  water. 


324  PHYSIOLOGICAL    CHEMISTRY. 

is  not  as  accurate  since  by  means  of  the  procedure  invohcd.  either 
alcohol  or  aldehyde  will  yield  a  precipitate  of  iodoform.  This  test  is 
especially  liable  to  lead  to  erroneous  deductions  when  urines  from 
the  advanced  stages  of  diabetes  are  under  examination,  because  of 
the  presence  of  alcohol  formed  from  the  sugar  through  fermentative 
processes.^ 

5.  Reynolds-Gunning  Test. — This  test  depends  upon  the  solu- 
bility of  mercuric  oxide  in  acetone  and  is  performed  as  follows:  To 
5  c.c.  of  the  urine  or  distillate  add  a  few  drops  of  mercuric  chloride, 
render  the  solution  alkaline  with  potassium  hydroxide  and  add  an 
equal  volume  of  95  per  cent  alcohol.  Shake  thoroughly  in  order  to 
bring  the  major  portion  of  the  mercuric  oxide  into  solution  and  filter. 
Render  the  clear  filtrate  faintly  acid  with  hydrochloric  acid  and  stratify 
some  ammonium  sulphide,  (NH^)2S,  upon  this  acid  solution.  At  the 
zone  of  contact  a  blackish-gray  ring  of  precipitated  mercuric  sulphide, 
HgS,  will  form.  Aldehyde  also  responds  to  this  test.  Aldehyde, 
however,  has  never  been  detected  in  the  urine  and  could  only  be 
present  in  this  instance  if  the  acidified  urine  was  distilled  too  far. 

6.  Taylor's  Test. — To  10  c.c.  of  the  urine  or  distillate  in  a  test- 
tube  add  a  few  drops  of  a  freshly  prepared  aqueous  solution  of  sodium 
nitroprusside  and  stratify  concentrated  ammonium  hydroxide  upon 
the  mixture.  The  production  of  a  magenta  color  at  the  point  of  con- 
tact indicates  the  presence  of  acetone  in  the  urine  or  distillate  under 
examination.  Normal  urine  yields  an  orange-red  color  when  subjected 
to  this  technique. 

CH3 
DIACETIC  ACID,  C  =  O 

CH^.COOH. 

Diacetic  or  acetoacetic  acid  occurs  in  the  urine  only  under  path- 
ological conditions  and  is  rarely  found  except  associated  with  acetone. 
It  is  formed  from  /?-oxybulyric  acid,  another  of  the  acetone  bodies,  and 
ujjon  decompfjsition  yields  acet(jne  and  carbon  dioxide.  Diaceturia 
occurs  ordinarily  under  the  same  conditions  as  the  ])alh()]ogical  ace- 
tonuria,  i.  e.,  in  fevers,  diabetes,  etc.  (see  p.  322).  If  \(ry  little  diacetic 
acid  is  formed  it  mav  all  be  Iransformed   inlo  acetone,  whereas  if  a 

'  Wclker  reports  ihc  [^rorluction  of  a  pink  or  red  color  during  tlie  iip|)liratiun  of  this 
test  to  the  distillates  from  pathological  urines  which  had  been  preserved  with  |)owdcred 
thymol.  He  found  the  color  to  be  due  to  an  iodothymol  < ompoiind  wliii  h  had  been 
previously  prepared  synthetically  by  Messinger  and  Vorlmaiin. 


URINE.  325 

larger  quantity  is  produced  both  acetone  and  diacetic  acid  may  be 
present  in  the  urine.  Diaceturia  is  most  frequently  observed  in  chil- 
dren, especially  accompanying  fevers  and  digestive  disorders;  it  is 
perhaps  less  frequently  observed  in  adults,  but  when  present,  particu- 
larly in  fevers  and  diabetes,  it  is  frequently  followed  by  fatal  coma. 

Diacetic  acid  is  a  colorless  liquid  which  is  miscible  with  water, 
alcohol,  and  ether,  in  all  proportions.  It  differs  from  acetone  in  giving 
a  violet-red  or  Bordeaux-red  color  with  a  dilute  solution  of  ferric 
chloride. 

Experiments. 

I.  Gerhardt's  Test. — To  5  c.c.  of  urine  in  a  test-tube  add  ferric 
chloride  solution,  drop  by  drop,  until  no  more  precipitate  forms!  In 
the  presence  of  diacetic  acid  a  Bordeaux-red  color  is  produced;  this 
color  may  be  somewhat  masked  by  the  precipitate  of  ferric  phosphate, 
in  which  case  the  fluid  should  be  filtered. 

A  positive  result  from  the  above  manipulation  simply  indicates 
the  possible  presence  of  diacetic  acid.  Before  making  a  final  decision 
regarding  the  presence  of  this  body  make  the  two  following  control 
experiments : 

{a)  Place  5  c.c.  of  urine  in  a  test-tube  and  boil  it  vigorously  for 
3-5  minutes.  Cool  the  tube  and,  with  the  boiled  urine,  make  the  test 
as  given  above.  As  has  been  already  stated,  diacetic  acid  yields 
acetone  upon  decomposition  and  acetone  does  not  give  a  Bordeaux- 
red  color  with  ferric  chloride.  By  boiling  as  indicated  above,  there- 
fore, any  diacetic  acid  present  would  be  decomposed  into  acetone 
and  carbon  dioxide  and  the  test  upon  the  resulting  fluid  would  be 
negative.  If  positive  the  color  is  due  to  the  presence  of  bodies  other 
than  diacetic  acid. 

ih)  Place  5  c.c.  of  urine  in  a  test-tube,  acidify  with  H2S0^,  to  free 
diacetic  acid  from  its  salts,  and  carefully  extract  the  mixture  with  ether 
by  shaking.  If  diacetic  acid  is  present  it  will  be  extracted  by  the 
ether.  Now  remove  the  ethereal  solution  and  add  to  it  an  equal 
volume  of  ferric  chloride;  diacetic  acid  is  indicated  by  the  production 
of  the  characteristic  Bordeaux-red  color.  This  color  disappears  spon- 
taneously in  24-48  hours.  Such  substances  as  antipyrin,  kairin, 
phenacetin,  salicylic  acid,  salicylates,  sodium  acetate,  thiocyanates, 
and  thallin  yield  a  similar  red  color  under  these  conditions,  but  when 
due  to  the  presence  of  any  of  these  substances  the  color  does  not  dis- 
appear spontaneously  but  may  remain  permanent  for  days.     Many 


326  PHYSIOLOGICAL    CHEMISTRY. 

of  these  disturbing  substances  are  soluble  in  benzene  or  chloroform 
and  may  be  removed  from  the  urine  by  this  means  before  extracting 
with  ether  as  above.  Diacetic  acid  is  insoluble  in  benzene  or 
chloroform. 

2.  Arnold-Lipliawsky  Reaction. — This  reaction  is  somewhat 
more  delicate  than  Gerhardt's  test  (i)  and  serves  to  detect  diacetic 
acid  when  present  in  the  proportion  of  1:25,000.  It  is  also  negative 
toward  acetone,  .J'-oxybutyric  acid  and  the  interfering  drugs  men- 
tioned as  causing  erroneous  deductions  in  the  application  of  Ger- 
hardt's test.  If  the  urine  under  examination  is  highly  pigmented 
it  should  be  partly  decolorized  by  means  of  animal  charcoal  before 
applying  the  test  as  indicated  below. 

Place  5  c.c.  of  the  urine  under  examination  and  an  equal  volume 
of  the  Arnold-Lipliawsky  reagent^  in  a  test-tube,  add  a  few  drops 
of  concentrated  ammonia  and  shake  the  tube  vigorously.  Note  the 
production  of  a  brick-red  color.  Take  1-2  c.c.  of  this  colored  solution, 
add  10-20  c.c.  of  hydrochloric  acid  (sp.  gr.  1.19),  3  c.c.  of  chloroform, 
and  2-4  drops  of  ferric  chloride  solution  and  carefully  mix  the  fluids 
without  shaking.  Diacetic  acid  is  indicated  by  the  chloroform  assuming 
a  \iolet  or  blue  color;  if  diacetic  acid  is  absent  the  color  may  be  yellow 
or  light  red. 

H    OHH 

,5-OXYBUTYRIC  ACID,     H-C-C-C-  CO  OH. 

H    H    H 

This  acid  does  not  occur  as  a  normal  constituent  of  urine  but  is 
found  only  under  pathological  conditions  and  then  always  in  con- 
junction with  either  acetone  or  diacetic  acid.  Either  of  these  bodies 
may  be  formed  from  /^J^-oxy butyric  acid  under  proj)cr  conditions.  It 
is  present  in  especially  large  amount  in  severe  cases  of  diabetes  and 
has  also  been  detected  in  digestive  disturbances,  continued  fevers, 
scurvy,  measles,  and  in  starvation.  It  is  prolxible  that,  in  man,  /?-oxy- 
butyric  acid,  in  common  with  acetone  and  diacetic  acid,  arises  prin- 
cipally from  the  Ijreaking  flown  of  fatty  tissues  williin  the  organism. 

'  This  reagent  consists  of  two  ficfmite  solutions  whicii  are  ordinarily  jjreserved  si'|)ai:ili-ly 
and  mixed  just  beff>re  using.     The  two  solutions  are  f)repared  as  follows: 

(a)  One  per  cent  aqueous  sf)lution  of  potassium  nitrite. 

(b)  One  gram  of  /»-amino-acetophenon  dissolverl  in  loo  c.c.  of  distilled  water  and 
enough  hydrochloric  acid  (about  2  c.c.)  added,  droj)  by  drop,  to  cause  lh(-  solution,  which 
is  at  first  yellow,  to  Ijecome  entirely  colorless.     An  excess  of  acid  must  be  avoided. 

Before  using,  a  and  b  are  mixed  in  the  ratio  i  :  2. 


URINE.  327 

The  condition  in  which  large  amounts  of  acetone  and  diacetic  acid, 
and  in  severe  cases  /?-oxybutyric  acid  also,  are  excreted  in  the  urine  is 
known  as  "acidosis."  In  diabetes  the  deranged  metabolic  conditions 
cause  the  production  of  great  quantities  of  these  substances  which 
lead  to  an  acid  intoxication  and  ultimately  to  diabetic  coma. 

Ordinarily  /J-oxybutyric  acid  is  an  odorless,  transparent  syrup, 
which  is  lasvorotatory  and  easily  soluble  in  water,  alcohol,  and  ether; 
it  may  be  obtained  in  crystalline  form. 

Experiments. 

I.  Black's  Reaction. — Inasmuch  as  the  urinary  pigments  as  well 
as  any  contained  sugar  or  diacetic  acid  will  interfere  with  the  delicacy 
of  this  test  when  applied  to  the  urine  directly  the  following  preliminary 
procedure  is  necessary:  Concentrate  10  c.c.  of  the  urine  under  exam- 
*  ination  to  one-third  or  one-fourth  of  its  original  volume  in  an  evaporat- 
ing dish  at  a  gentle  heat.  Acidify  the  residue  with  a  few  drops  of  con- 
centrated hydrochloric  acid,  add  sufficient  plaster  of  Paris  to  make  a 
thick  paste  and  allow  the  mixture  to  stand  until  it  begins  to  "set." 
It  should  now  be  stirred  and  broken  up  in  the  dish  by  means  of  a  stir- 
ring rod  with  a  blunt  end.  Extract  the  porous  meal  thus  produced 
twice  with  ether  by  stirring  and  decantation.  Any  /?-oxybutyric  acid 
present  will  be  extracted  by  the  ether.  Evaporate  the  ether  extract 
spontaneously  or  on  a  water-bath,  dissolve  the  residue  in  water,  and 
neutralize  it  with  barium  carbonate.  To  5  to  10  c.c.  of  this  neutral 
fluid  in  a  test-tube  add  two  to  three  drops  of  ordinary  commercial  acid 
hydrogen  peroxide.  Mix  by  shaking  and  add  a  few  drops  of  Black's 
reagent.^  Permit  the  tube  to  stand  and  note  the  gradual  development 
of  a  rose  color  which  increases  to  its  maximum  intensity  and  then 
gradually  fades.  ^ 

In  carrying  out  the  test  care  should  be  taken  to  see  that  the  solu- 
tion is  cold  and  approximately  neutral  and  that  a  large  excess  of  hydrogen 
peroxide  and  Black's  reagent  are  not  added.  In  case  but  little  /?-oxy- 
butyric  acid  is  present  the  color  will  fail  to  appear  or  will  be  but  tran- 
sitory if  the  oxidizing  agents  are  added  in  too  great  excess.  It  is  prefer- 
able to  add  a  few  drops  of  the  reagent  and  at  intervals  of  a  few  min- 
utes repeat  the  process  until  the  color  undergoes  no  further  increase 
in  intensity.  One  part  of  /5-oxybutyric  acid  in  10,000  parts  of  the  solu- 
tion may  be  detected  by  this  test. 

^  Made  by  dissolving  5  grams  of  ferric  chloride  and  o .  4  gram  of  ferrous  chloride 
in  100  c.c.  of  water. 

^  This  disappearance  of  color  is  due  to  the  further  oxidation  of  the  diacetic  acid. 


^26  PHYSIOLOGICAL    CHEMISTRY. 

2.  Polariscopic  Examination. — Subject  some  of  the  urine  (free 
from  protein)  to  the  ordinary  fermentation  test  (see  page  307).  This 
will  remove  dextrose  and  Itevulose,  which  would  interfere  with  the 
polariscopic  test.  Now  examine  the  fermented  tiuid  in  the  polariscopc 
and  if  it  is  laevorotatory  the  presence  of  ^5-oxybutyric  acid  is  indicated. 
This  test  is  not  absolutely  reliable,  however,  since  conjugate  glycu- 
ronates  are  also  laevorotatory  after  fermentation. 

3.  Kulz's  Test. — Evaporate  the  urine,  after  fermenting  it  as 
indicated  in  the  last  test,  to  a  syrup,  add  an  equal  volume  of  concen- 
trated sulphuric  acid,  and  distil  the  mixture  directly  without  cooling. 
Under  these  conditions  a-crotonic  acid  is  formed  and  is  present  in 
the  distillate.  Allow  the  distillate  to  cool  slowly  and  note  the  formation 
of  crystals  of  a-crotonic  acid  which  are  soluble  in  ether  and  melt  at 
72°  C.  In  case  very  slight  traces  of  /?-oxybutyric  acid  be  present  in 
the  urine  under  examination  the  amount  of  a-crotonic  acid  formed 
may  be  too  small  to  yield  a  crystalline  product.  In  this  event  the 
distillate  should  be  extracted  with  ether,  the  ethereal  extract  evaporated, 
and  the  residue  washed  with  water.  Under  these  conditions  the  im- 
purities will  be  removed  and  the  a-crotonic  acid  will  remain  behind 
as  a  residue.    The  melting-point  of  this  residue  may  then  be  determined. 

CONJUGATE   GLYCURONATES. 

Glycuronic  acid  does  not  occur  free  in  the  urine,  but  is  found,  for 
the  most  part,  in  combination  with  phenol.  Much  smaller  quantities 
are  excreted  in  combination  with  indoxyl  and  skatoxyl.  The  total 
content  of  conjugate  glycuronates  seldom  exceeds  0.004  per  cent 
under  normal  conditions.  The  output  may  be  very  greatly  increased 
as  the  result  of  the  administration  of  antipyrin,  borneol,  camphor, 
chloral,  menthol,  morphine,  naphthol,  turpentine,  etc.  The  glycu- 
ronates as  a  group  are  laevorotatory,  whereas  glycuronic  acid  is  dextro- 
rotatory. Most  of  the  glycuronates  reduce  alkaline  metallic  oxides 
and  so  introduce  an  error  in  the  examination  of  urine  for  sugar. 
Conjugate  glycuronates  often  occur  associated  with  dextrose  in  gly- 
cosuria, diabetes  mellitus,  and  in  some  other  disorders.  As  a  class 
the  glycuronates  are  non  fermental)Ie. 

Experiments. 

I.  Fermentation-Reduction  Test. — Test  the  urine  by  Fehling's 
test,  if  there  is  reduction  try  Barf(;ed's  test.  If  negative  this  indicates 
the  absence  of  monosaccharides.    A  negative  fermentation  test  would 


URINE.  329 

now  indicate  the  presence  of  conjugate  glycuronates  (or  lactose  in 
rare   cases)/ 

If  dextrose  is  present  in  the  urine  tested  for  glycuronates  the  urine 
must  first  be  subjected  to  a  polariscopic  examination,  then  fermented 
and  a  second  polariscopic  examination  made.  The  sugar  being 
dextrorotatory  and  fermentable  and  the  glycuronates  being  lasvoro- 
tatory  and  non-fermentable  the  second  polariscopic  test  will  show  a 
Isevorotation  indicative  of  conjugate  glycuronates. 

2.  Tollens'  Reaction. — Make  this  test  according  to  directions 
given  under  Pentoses,  below. 

PENTOSES. 

We  have  two  distinct  types  of  pentosuria,  i.  e.,  alimentary  pen- 
tosuria, resulting  from  the  ingestion  of  large  quantities  of  pentose- 
rich  vegetables  such  as  prunes,  cherries,  grapes,  or  plums,  and  fruit 
juices,  in  which  condition  the  pentoses  appear  only  temporarily  in  the 
urine;  and  the  chronic  form  of  pentosuria,  in  which  the  output  of  pen- 
toses bears  no  relation  whatever  to  the  quantity  and  nature  of  the 
pentose  content  of  the  food  eaten.  In  occurring  in  these  two  forms, 
pentosuria  resembles  glycosuria  (see  page  300),  but  it  is  definitely 
known  that  pentosuria  bears  no  relation  to  diabetes  mellitus  and  there 
is  no  generally  accepted  theory  to  account  for  the  occurrence  of  the 
chronic  form  of  pentosuria.  The  pentose  detected  most  frequently 
in  the  urine  is  arabinose,  the  inactive  form  generally  occurring  in 
chronic  pentosuria  and  the  Isevorotatory  variety  occurring  in  the 
alimentary  type  of  the  disorder. 

Experiments. 

1.  Tollens'  Reaction. — To  equal  volumes  of  urine  and  hydro- 
chloric acid  (sp.  gr.  1.09)  add  a  little  phloroglucin  and  heat  the  mix- 
ture on  a  boiling  water-bath.  Pentose,  galactose,  or  glycuronic  acid 
will  be  indicated  by  the  appearance  of  a  red  color.  To  differen- 
tiate between  these  bodies  examine  by  the  spectroscope  and  look  for 
the  absorption  band  between  D  and  E  given  by  pentoses  and  glycuronic 
acid,  and  then  differentiate  between  the  two  latter  bodies  by  the  melting- 
points  of  their  osazones. 

2.  Orcin  Test. — Place  equal  volumes  of  urine  and  hydrochloric 
acid  (sp.  gr.  1.09)  in  a  test-tube,  add  a  small  amount  of  orcin,  and 

'  If  necessary  to  differentiate  between  lactose  and  glycuronates  apply  the  mucic  acid 
test  (see  p.  40)  or  the  phenylhydrazine  reaction  (see  p.  23). 


33©  PHYSIOLOGICAL    CHEMISTRY. 

heat  the  mLxture  to  boiling.  Color  changes  from  red  through  reddish- 
blue  to  green  will  be  noted.  When  the  solution  becomes  green  it 
should  be  shaken  in  a  separator}'  funnel  with  a  little  amyl  alcohol, 
and  the  alcoholic  extract  examined  spectroscopically.  An  absorption 
band  between  C  and  D  will  be  observed. 

FAT. 

When  fat  finds  its  way  into  the  urine  through  a  lesion  which  brings 
some  portion  of  the  urinary  passages  into  communication  with  the 
lymphatic  system  a  condition  known  as  chyluria  is  established.  The 
turbid  or  milky  appearance  of  such  urine  is  due  to  its  content  of  chyle. 
This  disease  is  encountered  most  frequently  in  tropical  countries,  but 
is  not  entirely  unknown  in  more  temperate  climates.  Albumin  is  a 
constant  constituent  of  the  urine  in  chyluria.  Upon  shaking  a  chylous 
urine  with  ether  the  fat  is  dissolved  by  the  ether  and  the  urine  be- 
comes clearer  or  entirely  clear. 

H^MATOPORPHYRIN. 

Urine  containing  this  body  is  occasionally  met  with  in  various 
diseases,  but  more  frequently  after  the  use  of  quinine,  tetronal,  trional, 
and  especially  sulphonal.  Such  urines  ordinarily  possess  a  reddish 
tint,  the  depth  of  color  varying  greatly  under  different  conditions. 

Experiments. 

1.  Spectroscopic  Examination. — To  loo  c.c.  of  urine  add  about 
20  c.c.  of  a  ID  per  cent  solution  of  potassium  hydroxide  or  ammo- 
nium hydroxide.  The  precipitate  which  forms  consists  principally 
of  earthy  phosphates  to  which  the  hiemato])()r])hyrin  adheres  and  is 
carried  down.  Filter  off  the  precipitate,  wash  it  and  transfer  to  a  flask 
and  warm  with  alcohol  acidified  with  hydrochloric  acid.  By  this 
process  the  ha;mato]Kjrj;hyrin  is  dissolved  and  on  filtering  will  be  found 
in  the  filtrate  and  may  be  identified  by  means  of  the  si)ectroscope 
(see  page  202,  and  Absorption  Spectra,  Plate  llj. 

2.  Acetic  Acid  Test. — To  100  c.c.  of  urine  add  5  c.c.  of  glacial 
acetic  acid  and  allow  the  mixture  to  stand  4<S  hours.  Ihematopor- 
phyrin  deposits  in  the  U)vm  of  a  precipitate. 

LACTOSE. 

Lactose  is  rarely  found  in  the  urine  excej)t  as  it  is  excreted  by  women 
during  pregnancy,  during  the  nursing  period,  or  soon  after  weaning. 


URINE.  331 

It  is  rather  difficult  to  show  the  presence  of  lactose  in  the  urine 
in  a  satisfactory  manner,  since  the  formation  of  the  characteristic 
lactosazone  is  not  attended  with  any  great  measure  of  success  under 
these  conditions.  It  is,  however,  comparatively  easy  to  show  that 
it  is  not  dextrose,  for,  while  it  responds  to  reduction  tests,  it  does  not 
ferment  with  pure  yeast  and  does  not  give  a  dextrosazone.  An  abso- 
lutely conclusive  test,  of  course,  is  the  isolation  of  the  lactose  in  crystal- 
line form  (Fig.  75,  p.  215)  from  the  urine. 

On  oxidation  with  nitric  acid  lactose  and  galactose  yield  miicic 
acid.  This  test  is  frequently  used  in  urine  examination  to  differen- 
tiate lactose  and  galactose   from  other  reducing  sugars. 


Experiments. 

1.  Mucic  Acid  Test. — Treat  100  c.c.  of  the  urine  under  examina- 
tion with  20  c.c.^  of  concentrated  nitric  acid  and  evaporate  the  mix- 
ture in  a  broad,  shallow  glass  vessel,  upon  a  boiling  water-bath  until 
the  volume  of  the  solution  is  only  about  20  c.c.  At  this  point  the  fluid 
should  be  clear  and  a  fine  white  precipitate  of  mucic  acid  should 
separate.  If  the  percentage  of  lactose  in  the  urine  is  low  it  may  be 
necessary  to  cool  the  solution  and  permit  it  to  stand  for  some  time 
before  the  precipitate  will  form.  It  is  impossible  to  differentiate  between 
galactose  and  lactose  by  means  of  this  test,  but  the  reaction  does  serve 
to  differentiate  these  two  sugars  from  all  other  reducing  sugars.  A 
satisfactory  differentiation  between  lactose  and  galactose  may  be  made 
by  means  of  Barfoed's  test,  p.  30. 

2.  Rubner's  Test. — To  10  c.c.  of  urine  in  a  small  beaker  add 
some  plumbic  acetate,  in  substance,  heat  to  boiling,  and  add  NH^OH 
until  no  more  precipitate  is  dissolved.  In  the  presence  of  lactose 
a  brick-red  or  rose-red  color  develops,  whereas  dextrose  gives  a  coffee- 
brown  color,  maltose  a  light  yellow  color,  and  lagvulose  no  color  at  all 
under  the  same  conditions. 

3.  Compound  Test. — Try  the  phenylhydrazine  test,  the  fermen- 
tation test,  and  Barfoed's  test  according  to  directions  given  under 
Dextrose,  pages  23,  and  30.  If  these  are  negative,  try  Nylander's 
test,  page  29.  If  this  last  test  is  positive,  the  presence  of  lactose  is 
indicated. 


'  If  the  specific  gravity  of  the  urine  is  1020  or  over  it  is  necessary  to  use  25-35  c.c. 
of  nitric  acid.  Under  these  conditions  the  mixture  should  be  evaporated  until  the  remain- 
ing volume  is  approximately  equivalent  to  that  of  the  nitric  acid  added. 


T,;^2  PHYSIOLOGICAL    CHEMISTRY. 

GALACTOSE. 

Galactose  has  occasionally  been  detected  in  the  urine,  and  in  par- 
ticular in  that  of  nursing  infants  afHicted  with  a  deranged  digestive 
function.  Lactose  and  galactose  may  be  differentiated  from  other 
reducing  sugars  which  may  be  present  in  the  urine  by  means  of  the 
mucic  acid  test.  This  test  simply  consists  in  the  production  of  mucic 
acid  through  oxidation  of  the  sugar  with  nitric  acid. 

Experiments. 

1.  Mucic  Acid  Test. — Treat  loo  c.c.  of  the  urine  under  examin- 
ation with  20  c.c.^  of  concentrated  nitric  acid  and  evaporate  the  mix- 
ture in  a  broad,  shallow  glass  vessel,  upon  a  boiling  water-bath,  until 
the  volume  of  the  solution  is  only  about  20  c.c.  At  this  point  the 
fluid  should  be  clear  and  a  fine,  white  precipitate  of  mucic  acid  should 
separate.  If  the  percentage  of  galactose  present  in  the  urine  is  low 
it  may  be  necessary  to  cool  the  solution  and  permit  it  to  stand  for  some 
time  before  the  precipitate  will  form.  It  is  impossible  to  differentiate 
between  galactose  and  lactose  by  means  of  this  test,  but  the  reaction 
does  serve  to  differentiate  these  two  sugars  from  all  other  reducing 
sugars.  A  satisfactory  differentiation  between  galactose  and  lactose 
may  be  made  by  Barfoed's  test,  p.  30. 

2.  Tollens'  Reaction. — To  equal  \olumes  of  the  urine  and  hy- 
drochloric acid  (sp.  gr.  1.09)  add  a  little  ])hloroglucin  and  heat  the 
mixture  on  a  boiling  water-bath.  Galactose,  pentose,  and  glycu- 
ronic  acid  will  be  indicated  by  the  appearance  of  a  red  color.  Galac- 
tose may  be  differentiated  from  the  two  latter  substances  in  that 
its  solutions  exhibit  no  absor])tion  bands  upon  spectroscopical 
examination. 

L.EVULOSE. 

Diabetic  urine  frcqiK'nlly  ])ossesses  the  jjower  of  rotating  the 
plane  of  yjolarized  light  to  the  left,  thus  indicating  the  presence  of 
a  iievorotatory  substance.  This  laevorotation  is  sometimes  due  to  the 
presence  of  laevulose,  although  not  necessarily  confined  to  this  carbo- 
hydrate, since  conjugate  glycuronatcs  and  /'V-oxylnityric  acid,  two 
other  Itevorotatory  boflies,  are  frc(|iicntly  found  in  the  urine  of  diabetics. 
Ltevulose   is   in\ariably  accompanied    by   dextrose   in    diabetic    urine, 

'  If  the  s|jcrifi(;  grasity  of  the  urine  is  1020  or  over  it  is  necessary  to  use  25-35  c.c. 
of  nitric  acifl.  Under  these  ( onditions  the  mixture  should  l)e  eva|)oriited  until  the  remain- 
ing volume  is  ai>|)roximately  e'|uivalent  to  that  of  I  he  nitric  acid  added. 


URINE.  2>33 

but  lavulosuria  has  been  observed  as  a  separate  anomaly.  The  pres- 
ence of  laevulose  may  be  inferred  when  the  percentage  of  sugar,  as 
determined  by  the  titration  method,  is  greater  than  the  percentage 
indicated  by  the  polariscopic  examination. 

Experiments. 

1.  Borchardt's  Reaction. — To  about  5  c.c.  of  urine  in  a  test- 
tube  add  an  equal  volume  of  25  per  cent  hydrochloric  acid  and  a  few 
crystals  of  resorcin.  Heat  to  boiling  and  after  the  production  of  a  red 
color,  cool  the  tube  under  nmning  water  and  transfer  to  an  evaporat- 
ing dish  or  beaker.  Make  the  mixture  slightly  alkaline  with  solid 
potassium  hydroxide,  return  it  to  a  test-tube,  add  2-3  c.c.  of  acetic 
ether,  and  shake  the  tube  vigorously.  In  the  presence  of  laevulose  the 
acetic  ether  is  colored  yellow. 

The  only  urinary  constituents  which  interfere  with  the  test  are 
nitrites  and  indican  and  these  interfere  only  when  they  are  simul- 
taneously present.  Under  these  conditions,  the  urine  should  be  acidi- 
fied with  acetic  acid  and  heated  to  boiling  for  one  minute  to  remove 
the  nitrites.  In  case  the  indican  content  is  very  large,  it  will  impart  a 
blue  color  to  the  acetic  ether,  thus  masking  the  yellow  color  due  to 
laevulose.  When  such  urines  are  to  be  examined,  the  indican  should 
first  be  removed  by  Obermayer's  test  (see  p.  275).  The  chloroform 
should  then  be  discarded,  the  acid-urine  mixture  diluted  with  one- 
third  its  volume  of  water,  and  the  test  applied  as  described  above. 
The  urine  of  patients  who  have  ingested  santonin  or  rhubarb  respond 
to  the  test.  The  test  will  serve  to  detect  laevulose  when  present  in  a 
dilution  of  i  :  2000,  i.  e.,  0.05    per  cent. 

2.  Seliwanoff's  Reaction. — To  5  c.c.  of  Seliwanoff's  reagent^ 
in  a  test-tube  add  a  few  drops  of  the  urine  under  examination  and 
heat  the  mixture  to  boiling.  The  presence  of  laevulose  is  indicated 
by  the  production  of  a  red  color  and  the  separation  of  a  red  pre- 
cipitate. The  latter  may  be  dissolved  in  alcohol  to  which  it  will  impart 
a  striking  red  color. 

If  the  boiling  be  prolonged  a  similar  reaction  may  be  obtained 
with  urines  containing  dextrose. 

3.  Phenylhydrazine  Test. — Make  the  test  according  to  directions 
under  Dextrose,  3,  page  23. 

4.  Polariscopic  Examination. — A  simple  polariscopic  examina- 
tion, when  taken  in  connection  with  other  ordinary  tests,  will  fur- 

^  Seliwanoff's  reagent  may  be  prepared  by  dissolving  0.05  gram  of  resorcin  in  100  c.c. 
of  dilute  (i  :  2}  hydrochloric  acid. 


00- 


PHYSIOLOGICAL   CHEMISTRY. 


nish  the  requisite  data  regarding  the  presence  of  Isevulose,  provided 
laevulose  is  not  accompanied  by  other  laevorotatory  substances,  such  as 
conjugate  glycuronates  and  5-oxybutyric  acid. 

CHOH 

/\ 
HOHC       CHOH 
mosiTE,  I        II 

HOHC       CHOH 


CHOH 

Inosite  occasionally  occurs  in  the  urine  in  albuminuria,  diabetes 
mellitus,  and  diabetes  insipidus.  It  is  claimed  also  that  copious 
water-drinking  causes  this  substance  to  appear  in  the  urine.  Inosite 
was  at  one  time  considered  to  be  a  sugar  but  is  now  known  to  be  hexa- 
hydroxybenzene,  as  the  above  formula  indicates.  It  is  an  example 
of  a  non-carbohydrate  in  whose  molecule  the  H  and  O  are  present 
in  the  proportion  to  form  water.  In  other  words  it  has  the  formula 
of  the  hexoses,  i.  e.,  CgHjjOg.  Inosite  occurs  widely  distributed  in 
the  vegetable  kingdom,  and  because  of  this  fact  the  theory  has  been 
voiced  that  it  represents  one  of  the  first  stages  in  the  conversion  of  a 
carbohydrate  into  the  benzene  ring.  It  is  found  in  the  liver,  spleen, 
lungs,  brain,  kidneys,  suprarenal  capsules,  muscles,  leucocytes,  testes, 
and  urine  under  normal  conditions. 

Experiment. 

I.  Detection  of  Inosite. — Acidify  the  urine  with  concentrated 
nitric  acid  and  evaporate  nearly  to  dryness.  Add  a  few  drops  of 
ammonium  hydroxide  and  a  little  calcium  chloride  solution  to  the 
moist  residue  and  evaporate  the  mixture  to  dryness.  In  the  pres- 
ence of  inosite  (o.ooi  gram)  a  Ijright  red  color  is  obtained. 

LAIOSE. 

This  substance  is  occasionally  found  in  the  urine  in  severe  cases 
of  diabetes  mellitus.  By  some  investigators  laiose  is  classed  with 
the  sugars.  It  resembles  laevulose  in  that  it  has  the  ])n)])crty  of  reduc- 
ing certain  metallic  oxides  and  is  kevorotalory,  but  differs  from  luivu- 
lose  in  being  amorphous,  non-fermentable,  and  in  not  possessing  a  sweet 
taste. 


URINE.  ■  335 

MELANINS. 

These  pigments  never  occur  normally  in  the  urine,  but  are  present 
under  certain  pathological  conditions,  their  presence  being  especially 
associated  with  melanotic  tumors.  Ordinarily  the  freshly  passed 
urine  is  clear,  but  upon  exposure  to  the  air  the  color  deepens  and 
may  at  last  be  very  dark  brown  or  black  in  color.  The  pigment  is 
probably  present  in  the  form  of  a  chromogen  or  melanogen  and  upon 
coming  in  contact  with  the  air  oxidation  occurs,  causing  the  trans- 
formation of  the  melanogen  into  melanin  and  consequently  the  darken- 
ing of  the  urine. 

It  is  claimed  that  melanuria  is  proof  of  the  formation  of  a  vis- 
ceral melanotic  growth.  In  many  instances,  without  doubt,  urines 
rich  in  indican  have  been  wrongly  taken  as  diagnostic  proof  of  melan- 
uria. The  pigment  melanin  is  sometimes  mistaken  for  indigo  and 
melanogen  for  indican.  It  is  comparatively  easy  to  differentiate 
between  indigo  and  melanin  through  the  solubility  of  the  former  in 
chloroform. 

In  rare  cases  melanin  is  found  in  urinary  sediment  in  the  form 
of  fine  amorphous  granules. 

Experiments. 

1.  Zeller's  Test. — To  50  c.c.  of  urine  in  a  small  beaker  add  an 
equal  volume  of  bromine  water.  In  the  presence  of  melanin  a  yellow 
precipitate  will  form  and  will  gradually  darken  in  color,  ultimately 
becoming  black. 

2.  von  Jaksch-PoUak  Reaction. — Add  a  few  drops  of  ferric 
chloride  solution  to  10  c.c.  of  urine  in  a  test-tube  and  note  the  for- 
mation of  a  gray  color.  Upon  the  further  addition  of  the  chloride 
a  dark  precipitate  forms,  consisting  of  phosphates  and  adhering  melanin. 
An  excess  of  ferric  chloride  causes  the  precipitate  to  dissolve. 

This  is  the  most  satisfactory  test  for  the  identification  of  melanin 
in  the  urine. 

UROROSEIN. 

This  is  a  pigment  which  is  not  present  in  normal  urine  but  may 
be  detected  in  the  urine  of  various  diseases,  such  as  pulmonary  tuber- 
culosis, typhoid  fever,  nephritis,  and  stomach  disorders.  Urorosein,  in 
common  with  various  other  pigments,  does  not  occur  preformed  in  the 
urine,  but  is  present  in  the  form  of  a  chromogen,  which  is  transformed 
into  the  pigment  upon  treatment  with  a  mineral  acid. 


336  physiological  chemistry. 

Experiments. 

1.  Robin's  Reaction. — Acidify  10  c.c.  of  urine  with  about  15 
drops  of  concentrated  hydrochloric  acid.  Upon  allowing  the  acidi- 
fied urine  to  stand,  a  rose-red  color  will  appear  if  urorosein  is  present. 

2.  Nencki  and  Sieber's  Reaction. — To  100  c.c.  of  urine  in  a 
beaker  add  10  c.c.  of  2^  per  cent  sulphuric  acid.  Allow  the  acidi- 
fied urine  to  stand  and  note  the  appearance  of  a  rose-red  color.  The 
pigment  may  be  separated  by  extraction  with  amyl  alcohol. 

UNKNOWN    SUBSTANCES. 

Ehrlich's  Diazo  Reaction. — Place  eciual  volumes  of  urine  and 
Ehrlich's  diazobenzenesulphonic  acid  reagent^  in  a  test-tube,  mix 
thoroughly  by  shaking,  and  quickly  add  ammonium  hydroxide  in 
excess.  The  test  is  positive  if  both  the  liuid  and  the  foam  assume  a 
red  color.  If  the  tube  is  allowed  to  stand  a  precipitate  forms,  the 
upper  portion  of  which  exhibits  a  blue,  green,  greenish-black,  or  violet 
color.  Normal  urine  gives  a  brownish-yellow  reaction  with  the  above 
manipulation. 

The  exact  nature  of  the  substance  or  substances  upon  whose  pres- 
ence in  the  urine  this  reaction  depends  is  not  well  understood.  Some 
investigators  claim  that  a  positive  reaction  indicates  an  abnormal 
decomposition  of  protein  material,  whereas  others  assume  it  to  be 
due  to  an  increased  excretion  of  alloxyproteic  acid,  oxyproteic  acid, 
or  uroferric  acid. 

The  reaction  may  be  taken  as  a  metabolic  sym]jtom  of  certain  dis- 
orders, which  is  of  value  diagnostically  only  when  taken  in  connec- 
tion with  the  other  symptoms.  The  reaction  appears  principally 
in  the  urine  in  febrile  disorders  and  in  particular  in  the  urine  in  ty- 
phoid fever,  tuberculosis,  and  measles.  The  reaction  has  also  been 
obtained  in  the  urine  in  various  other  disorders  such  as  carcinoma, 
chronic  rheumatism,  di]jhthcria,  erysipelas,  y)leurisy,  ])iuumonia,  scarlet 
fever,  sypjhilis,  tyjjhus,  etc.  The  administration  of  alcohol,  chrysaro- 
bin.  creosote,  cresol,  dionin,  guaiacol,  heroin,  m()r])hine,  naj)hlhalene, 

'  Two  separate  solutions  should  be  prepared  and  mixed  in  ddinile  proijorlions  when 
needed  for  use. 

(a)  Five  grams  of  sfidium  nitrite  dissolved  in  i  liter  of  distilled  wulcr. 

(b)  Five  grams  of  sulphanilic  acid  and  50  c.c.  of  hydrochloric  acifl  in  r  liter  of  distilled 
water. 

S(jlutions  a  and  /;  should  be  preserved  in  well  stojjpererl  vessels  and  nii.xed  in  the  pro- 
portion I  :  .50  when  rer|uircfi.  Green  asserts  that  greater  delicacy  is  sc(  urc<l  by  mixing  the 
solutions  in  the  proportion  1:100.  The  sodium  nitrite  dclcrioiatcs  iijjon  slanfling  and 
becomes  unfit  for  use  in  the  course  of  a  few  weeks. 


URINE. 


337 


opium,  phenol,  tannic  acid,  etc.,  will  also  cause  the  urine  to  give  a 
positive  reaction. 

The  following  chemical  reactions  take  place  in  this  test: 

(a)  NaN02+HCl  =  HN02+NaCl. 

NH,  N 

/      '  /   \ 

(b)  C,H,  +HNO,  =  C6H,  N+2H,0. 

HSO3  SO3 

Sulphanilic  acid.  Diazo-benzenesulphonic  acid. 


CH.IPTER  XX. 

URINE:  ORGANIZED  AND  UNORGANIZED 

SEDIMENTS. 

The  data  obtained  from  carefully  conducted  microscopical  exam- 
inations of  the  sediment  of  certain  pathological  urines  are  of  very 
great  importance,  diagnostically.  Too  little  emphasis  is  sometimes 
placed  upon  the  value  of  such  findings. 


I 


Fig.  97. — The  Purdy  Electric  Centrifuge. 


Fig.  q8. — Sediment  Tube  for  the 
Purdy  Electric  Centrifuge. 


The  sedimentary  conslilucnls  may  be  divided  into  two  classes, 
i.  e.,  organized  and  unorganized.  The  sediment  is  ordinarily  collected 
for  examination  by  means  of  the  centrifuge  (Fig.  97,  above).  An 
older  mclhofl,  and  one  still  in  \ogue  in  some  (juarters,  is  the  so-called 
gravity  method,  'i'his  sim];ly  consists  in  placing  the  urine  in  a  conical 
glass  and  allowing  the  sediment  to  settle.  'The  collecti(in  of  the  sedi- 
ment by  means  of  the  centrifuge,  however,  is  much  preferable,  since 


URINE.  339 

the  process  of  sedimentation  may  be  accomplished  by  the  use  of  this 
instrument  in  a  few  minutes,  and  far  more  perfectly,  whereas  when 
the  other  method  is  used  it  is  frequently  necessary  to  allow  the  urine 
to  remain  in  the  conical  glass  12-24  hours  before  sufficient  sediment 
can  be  secured  for  the  microscopical  examination. 

(a)  Unorganized  Sediments. 

Ammonium  magnesium  phosphate  ("Triple  phosphate"). 

Calcium  oxalate. 

Calcium  carbonate. 

Calcium  phosphate. 

Calcium  sulphate. 

Uric  acid. 

Urates. 

Cystine. 

Cholesterol. 

Hippuric  acid. 

Leucine  and  tyrosine. 

Hsematoidin  and  bilirubin. 

Magnesium  phosphate. 

Indigo. 

Xanthine. 

Melanin. 

Ammonium  Magnesium  Phosphate   ("Triple  Phosphate"). — 

Crystals  of  "triple  phosphate"  are  a  characteristic  constituent  of  the 
sediment  when  alkaline  fermentation  of  the  urine  has  taken  place  either 
before  or  after  being  voided.  They  may  even  be  detected  in  amphoteric 
or  slightly  acid  urine  provided  the  ammonium  salts  are  present  in 
large  enough  quantity.  This  substance  may  occur  in  the  sediment 
in  two  forms,  i.  e.,  prisms  and  the  feathery  type.  The  prismatic  form 
of  crystals  (Fig.  96,  p.  296)  is  the  one  most  commonly  observed  in  the 
sediment;  the  feathery  form  (Fig.  96,  p.  296)  predominates  when  the 
urine  is  made  ammoniacal  with  ammonia. 

The  sediment  of  the  urine  in  such  disorders  as  are  accompanied 
by  a  retention  of  urine  in  the  lower  urinary  tract  contains  "triple 
phosphate"  crystals  as  a  characteristic  constituent.  The  crystals 
are  frequently  abundant  in  the  sediment  during  paraplegia,  chronic 
cystitis,  enlarged  prostate,  and  chronic  pyelitis. 

Calcium  Oxalate. — Calcium  oxalate  is  found  in  the  urine  in  the 
form  of  at  least  two  distinct  types  of  crystals,  i.  e.,  the  dumb-bell  type 


340  PHYSIOLOGICAL    CHEMISTRY. 

and  the  octahedral  type  (Fig.  99,  below).  Either  form  may  occur  in 
the  sediment  of  neutral,  alkaline,  or  acid  urine,  but  both  forms  are 
found  most  frequently  in  urine  having  an  acid  reaction.  Occasionally, 
in  alkaline  urine,  the  octahedral  form  is  confounded  with  "triple 
phosphate"  crystals.  They  may  be  differentiated  from  the  phosphate 
crystals  by  the  fact  that  they  are  insoluble  in  acetic  acid. 

The  presence  of  calcium  oxalate  in  the  urine  is  not  of  itself  a  sign 
of  any  abnormality,  since  it  is  a  constituent  of  normal  urine.     It  is 


^ 


^     ♦ 


Fig.  99. — CALcruM  Oxalate.     (Ogden.) 

increased  above  the  normal,  however,  in  such  pathological  conditions 
as  diabetes  mellitus,  in  organic  diseases  of  the  liver,  and  in  various  other 
conditions  which  arc  accompanied  by  a  derangement  of  digestion  or 
of  the  oxidation  mechanism,  such  as  occurs  in  certain  diseases  of  the 
heart  and  lungs. 

Calcium  Carbonate. — Calcium  carbonate  crystals  form  a  typical 
constituent  of  the  urine  of  herbivorous  animals.  They  occur  less 
frequently  in  human  urine.  The  reaction  of  urine  containing  these 
crystals  is  nearly  always  alkaline,  although  they  may  occur  in  ampho- 
teric or  in  slightly  acid  urine.  It  generally  crystallizes  in  the  form  of 
granules,  spherules,  or  dumb-bells  (Fig.  100,  p.  341).  The  crystals  of 
calcium  carbonate  may  be  differentiated  from  calcium  oxalate  by  the 
fact  that  they  dissolve  in  acetic  acid  with  the  evolution  of  carbon  dioxide 
gas. 

Calcium  Phosphate  (Stellar  Phosphate).— Calcium  ])hosi>liale 
may  occur  in  the  urine  in  three  forms,  i.  e.,  amorphous,  granular,  or 
crystalline.  The  crystals  of  calcium  phosphate  are  ordinarily  pointed, 
wedge-shaped  formations  which  may  occur  as  indi\i(hi;il  crystals,  or 
grouped  together  in  more  or  less  regularly  formed  rosettes  (Fig.  76, 
p.  220;.  Acid  sodium  urate  crystals  (Fig.  102,  p.  343)  are  o  ten  mis- 
taken for  crystals  oi  calcium  ])hos])hate.  We  may  differentiate  between 
these  two  crystalline  forms  by  the  fad  that  acetic   ac  id  will  readily  dis- 


URINE. 


341 


solve  the  phosphate,  whereas  the  urate  is  much  less  soluble  and  when 
finally  brought  into  solution  and  recrystallized  one  is  frequently  enabled 
to  identify  uric  acid  crystals  which  have  been  formed  from  the  acid 
urate  solution.  The  clinical  significance  of  the  occurrence  of  calcium 
phosphate  crystals  in  the  urinary  sediment  is  similar  to  that  of  "triple 
phosphate"  (see  page  296). 


Fig.  100. — Calcium  Carbonate. 


Calcium  Sulphate. — Crystals  of  calcium  sulphate  are  of  quite 
rare  occurrence  in  the  sediment  of  urine.  Their  presence  seems  to 
be  limited  in  general  to  urines  which  are  of  a  decided  acid  reaction. 
Ordinarily  it  crystallizes  in  the  form  of  long,  thin,  colorless  needles 
or  prisms  (Fig.  95,  page  292)  which  may  be  mistaken  for  calcium  phos- 
phate crystals.  There  need  be  no  confusion  in  this  respect,  however, 
since  the  sulphate  crystals  are  insoluble  in  acetic  acid,  which  reagent 
readily  dissolves  the  phosphate.  As  far  as  is  known  their  occurrence 
as  a  constituent  of  urinary  sediment  is  of  very  little  clinical  significance. 

Uric  Acid. — Uric  acid  forms  a  very  common  constituent  of  the 
sediment  of  urines  which  are  acid  in  reaction.  It  occurs  in  more  varied 
forms  than  any  of  the  other  crystalline  sediments  (Plate  V,  opposite 
page  267,  and  Fig.  loi,  page  342),  some  of  the  more  common  varieties 
of  crystals  being  rhombic  prisms,  wedges,  dumb-bells,  whetstones, 
prismatic  rosettes,  irregular  or  hexagonal  plates,  etc.  Crystals  of  pure 
uric  acid  are  always  colorless  (Fig.  89,  page  269),  but  the  form  occurring 
in  urinary  sediments  is  impure  and  under  the  microscope  appears  pig- 
mented, the  depth  of  color  varying  from  light  yellow  to  a  dark  reddish- 
brown  according  to  the  size  and  form  of  the  crystal. 


342 


PHYSIOLOGICAL   CHEMISTRY. 


The  presence  of  a  considerable  uric  acid  sediment  does  not,  of 
necessity,  indicate  a  pathological  condition  or  a  urine  of  increased  uric 
acid  content,  since  this  substance  very  often  occurs  as  a  sediment  in 
urines  whose  uric  acid  content  is  diminished  from  the  normal  merely 
as  a  result  of  changes  in  reaction,  etc.  Pathologically,  uric  acid  sedi- 
ments occur  in  gout,  acute  febrile  conditions,  chronic  interstitial  neph- 
ritis, etc.  If  the  microscopical  examination  is  not  conclusive,  uric  acid 
mav  be  differentiated  from  other  crystalline  urinary  sediments  from 


Fig.  ioi. — ^Various  Forms  of  Uric  Acid. 
I,  Rhombic  plates;  2,  whetstone  forms;  3,  3,  quadrate  forms;  4,  5,  prolonged  into 
points;  6,  8,  rosettes;  7,  pointed  bundles;  9,  barrel  forms  precipitated  by  adding  hydro- 
chloric acid  to  urine. 


the  fact  that  it  is  soluble  in  alkalis,  alkali  carbonates,  boiling  glycerol, 
concentrated  sulphuric  acid,  and  in  certain  organic  bases  such  as  ethyl- 
amine  and  piperidin.  It  also  responds  to  the  murexide  test  (see  page 
269),  Schiflf's  reaction  (see  page  269)  and  to  Moreignc's  reaction  (see 
p.  269). 

Urates. — The  urate  sedimcnl  may  consist  of  a  mixture  of  the 
urates  of  ammonium,  calcium,  magnesium,  potassium,  and  sodium. 
The  ammonium  urate  may  occur  in  neutral,  alkaline,  or  acid  urine, 
whereas  the  other  forms  of  urates  are  confined  to  the  sech'ments  of  acid 
urines.  Sodium  urate  occurs  in  sediments  more  abundanlly  than  the 
other  urates.  The  urates  of  calcium,  magnesium,  and  ])olassium  are 
amorphous  in  character,  whereas  the  urate  of  ammonium  is  crystalline. 
Sodium  urate  may  be  cither  amorphous  or  crystalline.    When  crystal- 


PLATE  \'I. 


Ammonium  Urate,  siiowiNr;  Sphkrulks  and  Thorn-aim'i.k-siiai'kd  C'kysiai.s. 
(I'Vom  (h/i^ni,  aflcr  I'cyer.) 


URINE. 


343 


line  it  forms  groups  of  fan-shaped  clusters  or  colorless  prismatic 
needles  (Fig.  102,  below).  Ammonium  urate  is  ordinarily  present 
in  the  sediment  in  the  burr-like  form  of  the  "thorn-apple"  crystal, 
i.  e.,  yellow  or  reddish-brown  spheres,  covered  with  sharp  spicules 
or  prisms   (Plate  VI,  opposite).     The  urates  are  all  soluble  in  hydro- 


FiG.  102. — Acid  Sodium  Urate. 

chloric  acid  or  acetic  acid  and  their  acid  solutions  yield  crystals  of 
uric  acid  upon  standing.  They  also  respond  to  the  murexide  test. 
The  clinical  significance  of  urate  sediments  is  very  similar  to  that  of 
uric  acid.  A  considerable  sediment  of  amorphous  urates  does  not 
necessarily  indicate  a  high  uric  acid  content,  but  ordinarily  signifies  a 
concentrated  urine  having  a  very  strong  acidity. 


/ 

• 


Fig.  103. — Cystine.     (Ogden.) 


Cystine. — Cystine  is  one  of  the  rarer  of  the  crystalline  urinary 
sediments.  It  has  been  claimed  that  it  occurs  more  often  in  the  urine 
of  men  than  of  women.  Cystine  crystallizes  in  the  form  of  thin,  color- 
less, hexagonal  plates  (Fig.  24,  p.  71,  and  Fig.  103,  above)  which  are 
insoluble  in  water,  alcohol,  and  acetic  acid,  and  soluble  in  minerals  acids^ 


344  PHYSIOLOGICAL    CHEMISTRY. 

alkalis,  and  especially  in  ammonia.  Cystine  may  be  identified  by 
burning  it  upon  platinum  foil,  under  which  condition  it  does  not  melt 
but  yields  a  bluish-green  flame. 

Cholesterol. — Cholesterol  crystals  have  been  but  rarely  detected 
in  urinary  sediments.  When  present  they  probably  arise  from  a 
pathological  condition  of  some  portion  of  the  urinary  tract.  Crystals 
of  cholesterol  have  been  found  in  the  sediment  in  cystitis,  pyelitis,  chy- 
luria,  and  nephritis.  Ordinarily  it  crystallizes  in  large  regular  and 
irregular  colorless,  transparent  plates,  some  of  which  possess  notched 
corners  (Fig.  42,  page  155).  Frequently,  instead  of  occurring  in  the 
sediment,  it  is  found  in  the  form  of  a  film  on  the  surface  of  the  urine. 
Hippuric  Acid. — This  is  one  of  the  rarer  sediments  of  human 
urine.  It  deposits  under  conditions  similar  to  those  which  govern  the 
formation  of  uric  acid  sediments.  The  crystals,  which  are  colorless 
needles  or  prisms  (Fig.  92,  page  276)  when  pure,  are  invariably  pig- 
mented in  a  manner  similar  to  the  uric  acid  crystals  when  observed  in 
urinary  sediment  and  because  of  this  fact  are  frecjuently  confounded 
with  the  rarer  forms  of  uric  acid.  Hippuric  acid  may  be  dift'erentiated 
from  uric  acid  from  the  fact  that  it  does  not  respond  to  the  murexide 
test  and  is  much  more  soluble  in  water  and  in  ether.  The  detection  of 
crystals  of  hippuric  acid  in  the  urine  has  very  little  clinical  significance, 
since  its  presence  in   the  sediment  depends  in  most  instances  very 

greatly  upon  the  nature  of  the  diet. 
It  is  yjarticularly  prone  to  occur  in 
the  sediment  after  the  ingestion  of 
certain  fruits  as  well  as  after  the  inges- 
tion of  benzoic  acid  (see  page  276). 

Leucine  and  Tyrosine. — Leucine 
and  tyrosine  ha\'e  fre(|ucntly  been  de- 
tected in  the  urine,  either  in  solution 
d  or  as  a  sediment.     Neither  of  them 

iMG.  104.— Crystals  of  Lmpurk        occurs  in   the  urinc  ordinarily  except 
Leucine.     {Ogden.)  ■'        . 

in    association    with   the   other,    *.  e., 

whenever  leucine  is  detected  it  is  more  than  probable  that  tyrosine 
accompanies  it.  They  have  l)een  found  ])ath()l()girally  in  liie  urine 
in  acute  yelhnv  atroy)hy  of  the  liver,  in  acute  ])hos])h()rus  jjoisoning, 
in  cirrhosis  of  the  liver,  in  severe  cases  of  tyjjhoid  fever  and  smallpox, 
and  in  leukaemia.  In  urinary  sediments  leucine  ordinarily  crystallizes 
in  characteristic  sjiherical  masses  which  show  both  radial  and  concen- 
tric striations  and  are  highly  refractive  (Fig.  104,  above).  Some  inves- 
tigators claim  that  these  crystals  which  are  ordinarily  called  leucine 


w 


URINE.  345 

are,  in  reality,  generally  urates.  For  the  crystalline  form  of  pure  leucine 
obtained  as  a  decomposition  product  of  protein  see  Fig.  26,  p.  75. 
Tyrosine  crystallizes  in  urinary  sediments  in  the  well-known  sheaf  or 
tuft  formation  (Fig.  23,  p.  71).  For  other  tests  on  leucine  and  tyrosine 
see  pages  81  and  82. 

Hsematoidin  and  Bilirubin. — There  are  divergent  opinions 
regarding  the  occurrence  of  these  bodies  in  urinary  sediment.  Each 
of  them  crystallizes  in  the  form  of  tufts  of  small  needles  or  in  the  form 
of  small  plates  which  are  ordinarily  yellowish-red  in  color  (Fig.  41,  p. 
150).  Because  of  the  fact  that  the  crystalline  form  of  the  two  sub- 
stances is  identical  many  investigators  claim  them  to  be  one  and  the 
same  body.  Other  investigators  claim,  that  while  the  crystalline  form 
is  the  same  in  each  case,  there  are  certain  chemical  differences  which 
may  be  brought  out  very  strikingly  by  properly  testing.  For  in- 
stance, it  has  been  claimed  that  hsematoidin  may  be  differentiated 
from  bilirubin  through  the  fact  that  it  gives  a  momentary  color  reaction 
(blue)  when  nitric  acid  is  brought  in  contact  with  it,  and,  further,  that 
it  is  not  dissolved  on  treatment  with  ether  or  potassium  hydroxide. 
Pathologically,  typical  crystals  of  haematoidin  or  bilirubin  have  been 
found  in  the  urinary  sediment  in  jaundice,  acute  yellow  atrophy  of 
the  liver,  carcinoma  of  the  liver,  cirrhosis  of  the  liver,  and  in  phosphorus 
poisoning,  typhoid  fever,  and  scarlatina. 

Magnesium  Phosphate. — Magnesium  phosphate  crystals  occur 
rather  infrequently  in  the  sediment  of  urine  which  is  neutral,  alkaline, 
ox  feebly  acid  in  reaction.  It  ordinarily  crystallizes  in  elongated,  highly 
refractive,  rhombic  plates  which  are  soluble  in  acetic  acid. 

Indigo. — Indigo  crystals  are  frequently  found  in  urine  which  has 
undergone  alkaline  fermentation.  They  result  from  the  breaking 
down  of  indoxyl-sulphates  or  indoxyl-glycuronates.  Ordinarily  indigo 
deposits  as  dark  blue  stellate  needles  or  occurs  as  amorphous  particles 
or  broken  fragments.  These  crystalline  or  amorphous  forms  may  occur 
in  the  sediment  or  may  form  a  blue  film  on  the  surface  of  the  urine. 
Indigo  crystals  generally  occur  in  urine  which  is  alkaline  in  reaction, 
but  they  have  been  detected  in  acid  urine. 

Xanthine. — Xanthine  is  a  constituent  of  normal  urine  but  is  found 
in  the  sediment  in  crystalline  form  very  infrequently,  and  then  only  in 
pathological  urine.  When  present  in  the  sediment  xanthine  generally 
occurs  in  the  form  of  whetstone-shaped  crystals  somewhat  similar  in 
form  to  the  whetstone  variety  of  uric  acid  crystal.  They  may  be  differen- 
tiated from  uric  acid  by  the  great  ease  with  which  they  may  be  brought 
into  solution  in  dilute  ammonia   and   on   applying   heat.     Xanthine 


346  PHYSIOLOGICAL   CHEMISTRY. 

may  also  form  urinary  calculi.     The  clinical  significance  of  xanthine 
in  urinary  sediment  is  not  well  understood. 

Melanin. — ^Melanin  is  an  extremely  rare  constituent  of  urinary 
sediments.  Ordinarily  in  melanuria  the  melanin  remains  in  solution; 
if  it  separates  it  is  generally  held  in  suspension  as  fine  amorphous 
granules. 

(b)  Organized  Sediments. 

Epithelial  cells. 

Pus  cells. 

^  Hyaline. 
Granular. 
Epithelial. 

Casts.      Blood. 
Fatty. 
Waxy. 
I  Pus. 

Cylindroids. 

Erythrocytes. 

Spermatozoa. 

Urethral  filaments. 

Tissue  debris. 

Animal  parasites. 

Micro-organisms. 

Fibrin. 

Foreign  substances  due  to  c(jntamination. 

Epithelial  Cells. — The  detection  of  a  certain  number  of  these  cells 
in  urinary  sediment  is  not,  of  itself,  a  pathological  sign,  since  they  occur 
in  normal  urine.  However,  in  certain  pathological  conditions  they 
are  greatly  increased  in  number,  and  since  different  areas  of  the  urinary 
tract  are  lined  with  different  forms  of  epithelial  cells,  it  becomes  neces- 
sary, when  examining  urinary  sediments,  to  note  not  only  the  relative 
number  of  such  cells,  but  at  the  same  time  to  carefully  oljserve  the  shape 
of  the  various  individuals  in  order  to  determine,  as  far  as  possible, 
from  what  portion  of  the  tract  they  have  been  derived.  Since  the 
different  layers  of  the  epithelial  lining  are  composed  of  cells  different 
in  form  from  those  of  the  associated  layers,  it  is  evident  that  a  careful 
microscopical  examination  of  these  cells  may  tell  us  the  ])arlicular  layer 
which  is  being  desrjuamated.  It  is  fref|uently  a  most  diflicult  under- 
taking, however,  to  make  a  clear  differentiation  between  the  various 


URINE. 


347 


forms  of  epithelial  cells  present  in  the  sediment.  If  skilfully  done, 
such  a  microscopical  differentiation  may  prove  to  be  of  very  great 
diagnostic  aid. 

The  principal  forms  of  epithelial  cells  met  with  in  urinary  sediments 
are  shown  in  Fig.  lo^,  below. 


Fig.  105. — Epithelium  from  Different  Areas  of  the  Urinary  Tract. 
a,  Leucocyte  (for  comparison);  h,  renal  cells;  c,  superficial  pelvic  cells;  d,  deep  peh'ic 
cells;  e,  cells  from  calices;/,  cells  from  ureter;  g,  g,  g,  g,  g,  squamous  epithelium  from  the 
bladder;  h,  h,  neck-of-bladder  cells;  i,  epithelium  from  prostatic  urethra;  k,  urethral  cells; 
/,  I,  scaly  epithelium;  m,  m',  cells  from  seminal  passages;  n,  compound  granule  cells;  o 
fatty  renal  cell.     (Ogden.) 


Pus  Cells. — ^Pus  corpuscles  or  leucocytes  are  present  in  extremely 
small  numbers  in  normal  urine.  Any  considerable  increase  in  the 
number,  however,  ordinarily  denotes  a  pathological  condition,  gener- 
ally an  acute  or  chronic  inflammatory  condition  of  some  portion  of 
the  urinary  tract.  The  sudden  appearance  of  a  large  amount  of  pus 
in  a  sediment  denotes  the  opening  of  an  abscess  into  the  urinary  tract. 
Other  form  elements,  such  as  epithelial  cells,  casts,  etc.,  ordinarily 
accompany  pus  corpuscles  in  urinary  sediment  and  a  careful  examina- 
tion of  these  associated  elements  is  necessary  in  order  to  form  a  correct 
diagnosis  as  to  the  origin  of  the  pus.  Protein  is  always  present  in 
urine  which  contains  pus. 

The  appearance  w^hich  pus  corpuscles  exhibit  under  the  microscope 
depends  greatly  upon  the  reaction  of  the  urine  containing  them.  In 
acid  urine  they  generally  present  the  appearance  of  round,  colorless 
cells  composed  of  refractive,  granular  protoplasm,  and  may  frequently 
exhibit  amoeboid  movements,  especially  if  the  slide  containing  them 


348 


PHYSIOLOGICAL    CHEMISTRY. 


be  warmed  slightly.  They  are  nucleated  (one  or  more  nuclei),  the 
nuclei  being  clearly  visible  only  upon  treating  the  cells  with  water, 
acetic  acid,  or  some  other  suitable  reagent.  In  urine  which  has  a 
decided  alkaline  reaction,  on  the  other  hand,  the  pus  corpuscles  are 
often  greatly  degenerated.  They  may  be  seen  as  swollen,  transparent 
cells,  which  exhibit  no  granular  structure  and  as  the  process  of  degenera- 
tion continues  the  cell  outline  ceases  to  be  visible,  the  nuclei  fade,  and 
finally  only  a  mass  of  debris  containing  isolated  nuclei  and  an  occasional 
cell  remains. 


Fig.  io6. — Pus  Corpuscles.     (.After  Ultzviann.) 

I,  Normal;  2,  showing  amoeboid  movements;  3,  nuclei  rendered  distinct  by  acetic  acid; 

4,   as  observed  in  chronic   pyelitis;   5,   swollen   by  amniunium   (arbonatc. 


It  is  frequently  rather  difficull  to  make  a  (lilTiTenliation  between 
pus  corpuscles  and  certain  types  of  epithelial  cells  which  are  similar 
in  form.  Such  confusion  may  be  avoided  by  the  addition  of  iodine 
solution  (I  in  KI),  a  reagent  which  stains  the  pus  corpuscles  a  dec]3 
mahogany-brown  and  transmits  to  the  epithelial  cells  a  light  yellow 
tint.  The  test  j)ro[josed  by  X'ilali  often  gives  \ery  satisfactory  results. 
This  simply  consists  in  acidifying  the  urine  (if  alkaline)  with  acetic  acid, 
then  filtering,  and  treating  the  sediment  on  the  filter  paper  with  freshly 
prepared  tincture  of  guaiac.  The  presence  of  pus  in  the  sediment  is 
indicated  if  a  hhie  color  is  observed.  T^arge  numbers  of  pus  corpuscles 
are  present  in  the  urinary  sediment  in  gonorrh(x;a,  leucorrha-a,  chronic 
pyelitis,  and  in  abscess  of  the  kidney. 

Casts.  These  arc  cylindrical  formations,  which  originate  in  the 
uriniferous  tubules  and  are  forced  out  by  the  pressure  of  the  urine. 


URINE. 


349 


They  vary  greatly  in  size,  but  in  nearly  every  instance  they  possess 
parallel  sides  and  rounded  ends.  The  finding  of  casts  in  the  urine  is 
very  important  because  of  the  fact  that  they  generally  indicate  some 
kidney  disorder;  if  albumin  accompanies  the  casts  the  indication  is 
much  accentuated.  Casts  have  been  classified  according  to  their 
microscopical  characteristics  as  follows:  (a)  Hyaline,  (6)  granular, 
(c)  epithelial,  {d)  blood,  {e)  fatty,  (/)  waxy,  {g)  pus. 

{a)  Hyaline  Casts. — These  are  composed  of  a  basic  material  which 
is  transparent,  homogeneous,  and  very  light  in  color  (Fig.  107,  below). 


Fig.  107. — Hyaline  Casts. 
One  cast  is  impregnated  with  four  renal  cells. 


In  fact,  chiefly  because  of  these  physical  properties,  they  are  the  most 
difficult  form  of  renal  casts  to  detect  under  the  microscope.  Frequentlv 
such  casts  are  impregnated  with  deposits  of  various  forms,  such  as 
erythrocytes,  epithelial  cells,  fat  globules,  etc.,  thus  rendering  the  form 
of  the  cast  more  plainly  visible.  Staining  is  often  resorted  to  in  order 
to  render  the  shape  and  character  of  the  cast  more  easily  determined. 
Ordinary  iodine  solution  (I  in  KI)  may  be  used  in  this  connection; 
many  of  the  aniline  dyes  are  also  in  common  use  for  this  purpose,  e.  g., 
gentian- violet,  Bismarck-brown,  methylene-blue,  fuchsin,  and  eosin. 
Generally,  but  not  always,  albumin  is  present  in  urine  containing  hya- 
line casts.     Hyaline  casts  are  common  to  all  kidney  disorders,  but 


350 


PHYSIOLOGICAL   CHEMISTRY. 


occur  particularly  in  the  earliest  and  recovering  stages  of  parenchy- 
matous nephritis  and  interstitial  nephritis. 

(b)  Granular  Casts. — The  common  hyaline  material  is  ordinarily 
the  basic  substance  of  this  form  of  cast.  The  granular  material  gener- 
ally consists  of  albumin,  epithelial  cells,  fat,  or  disintegrated  erythro- 
cytes or  leucocytes,  the  character  of  the  cast  varying  according  to  the 
nature  and  size  of  the  granules  (Fig.  io8,below,  and  Fig.  109,  page 
351).  Thus  we  have  casts  of  this  general  type  classified  as  Jinely 
granular  and  coarsely  granular  casts.     Granular  casts,  and  in  particular 


Fig.  108. — GR.'VNruLAR  Casts,     (.\fter  Peyer.) 

the  finely  granular  types,  occur  in  the  sediment  in  practically  every 
kidney  disorder  but  are  probably  especially  characteristic  of  the  sedi- 
ment in  inflammatory  disorders. 

(c)  Epithelial  Casts. — These  are  casts  bearing  upon  their  surface  epi- 
thelial cells  from  the  lining  of  the  urinifcrous  tubules  (Fig.  no,  p.  351). 
The  basic  material  of  this  form  of  cast  may  be  hyaline  or  granular  in 
nature.  Epithelial  casts  are  jjarticularly  abundant  in  the  urinary  sedi- 
ment in  acute  nejjhritis. 

(d)  Blood  Casts. — Casts  of  this  ty])C  may  consist  of  erythrocytes 
borne  ujKjn  a  hyaline  or  a  fibrinous  basis  (Fig.  iir,  ]).  351).  'J'he 
occurrence  of  such  casts  in  the  urinary  sediment  denotes  renal  hemor- 
rhage and  they  are  considered  to  be  especially  characteristic  of  acute 
difTuse  nephritis  and  acute  congestion  of  the  kidney. 


URINE. 


351 


{e)  Fatty  Casts. — Fatty  casts  may  be  formed  by  the  deposition  of 
fat  globules  or  crystals  of  fatty  acid  upon  the  surface  of  a  hyaline  or 
granular  cast  (Fig.  112,  p.  352).     In  order  to  constitute  a  true  fatty 


Fig.  109. — Granular  Casts. 
a,  Finely  granular;  b,  coarsely  granular. 


Fig.  no. — Epithelial  Casts. 


cast  the  deposited  material  must  cover  the  greater  part  of  the  surface 
area  of  the  cast.  The  presence  of  fatty  casts  in  urinary  sediment  indi- 
cates fatty  degeneration  of  the  kidney;  such  casts  are  particularly 
characteristic  of  subacute  and  chronic  inflammations  of  the  kidney. 


Fig.  hi. — Blood,  Pus,  Hyaline  and  Epithelial  Casts. 
a,  Blood  casts;  h,  pus  cast;  c,  hyaline  cast  impregnated  with  renal  cells;  d,  epithelial  casts. 


352 


PHYSIOLOGICAL    CHEMISTRY. 


Fig.  112.— Fatty  Casts.     (After  i^vc''.) 


Vic.  ii.}.     Fatty  anu  Waxy  Casts. 
a,  Patty  casts;  /;,  waxy  casts. 


URINE.  353 

(/)  Waxy  Casts. — These  casts  possess  a  basic  substance  similar  to 
that  which  enters  into  the  foundation  of  the  hyahne  form  of  cast.  In 
common  with  the  hyahne  type  they  are  colorless,  refractive  bodies,  but 
differ  from  this  form  of  cast  in  being,  in  general,  of  greater  length  and 
diameter  and  possessing  sharper  outlines  and  a  light  yellow  color  (Fig. 
113,  p.  352).  Such  casts  occur  in  several  forms  of  nephritis,  but  do 
not  appear  to  characterize  any  particular  type  of  the  disorder  except 
amyloid  disease,  in  which  they  are  rather  common. 

(g)  Pus  Casts. — Casts  whose  surface  is  covered  with  pus  cells  or 
leucocytes  are  termed  pus  casts  (Fig.  iii,  p.  351).  They  are  frequently 
mistaken  for  epithelial  casts.  The  differentiation  between  these  two 
types  is  made  very  simple,  however,  by  treating  the  cast  with  acetic 
acid  which  causes  the  nuclei  of  the  leucocytes  to  become  plainly  visible. 
The  true  pus  cast  is  quite  rare  and  indicates  renal  suppuration. 


Fig.  114. — Cylindroids.     (After  Pfver.) 

Cylindroids. — These  formations  may  occur  in  normal  or  patho- 
logical urine  and  have  no  particular  clinical  significance.  They  are 
frequently  mistaken  for  true  casts,  especially  the  hyaline  type,  but 
they  are  ordinarily  flat  in  structure  with  a  rather  smaller  diameter 
than  casts,  may  possess  forked  or  branching  ends,  and  are  not  com- 
posed of  homogeneous  material  as  are  the  hyaline  casts.  Such  "false 
casts"  may  become  coated  with  urates,  in  which  event  they  appear 


354 


PHYSIOLOGICAL  CHEMISTRY. 


granular  in  structure.    The  basic  substance  of  cylindroids  is  often  the 
nucleoprotein  of  the  urine  (see  Fig.  114,  page  353). 

Erythrocytes. — These  form  elements  are  present  in  the  urinary 
sediment  in  various  diseases.  They  may  appear  as  the  normal  bicon- 
cave, yellow  erythrocyte  (Plate  I\',  opposite  page  180)  or  may  exhibit 
certain  modifications  in  form,  such  as  the  crenated  type  (Fig.  115, 
below)  which  is  often  seen  in  concentrated  urine.  Under  different 
conditions  they  may  become  swollen  sufficiently  to  entirely  erase  the 
biconcave  appearance  and  may  even  occur  in  the  form  of  colorless 


Fig.  115. — Crenated  Erythrocytes. 


spheres  having  a  smaller  diameter  than  the  original  disc-shaped  cor- 
puscles. Erythrocytes  are  found  in  urinary  sediment  in  hemorrhage 
of  the  kidney  or  of  the  urinary  tract,  in  traumatic  hemorrhage,  hemor- 
rhage from  congestion,  and  in  hemorrhagic  diathesis. 

Spermatozoa. — Spermatozoa  may  be  detected  in  the  urinary  sedi- 
ment in  diseases  of  the  genital  organs,  as  well  as  after  coitus,  nocturnal 
emissions,  epileptic,  and  other  convulsive  attacks,  and  sometimes  in 
severe  febrile  disorders,  especially  in  typhoid  fever.  In  form  they 
consist  of  an  oval  body,  to  which  is  attached  a  long,  delicate  tail  (Fig. 
116,  p.  355).  Upon  examination  they  may  show  motility  or  may  be 
motionless. 

Urethral  Filaments. — These  are  peculiar  thread-like  bodies 
which  are  sometimes  found  in  urinary  sediment.  They  may  occa- 
sionally be  detected  in  normal  urine  and  pathologically  are  found  in 
the  sediment  in  acute  and  chronic  gonorrhoea  and  in  urethrorrha-a. 


URINE. 


355 


The  ground-substance  of  these  urethral  filaments  is,  in  part  at  least, 
similar  to  that  of  the  cylindroids  (see  page  353).  The  urine  first 
voided  in  the  morning  is  best  adapted  for  the  examination  for  fila- 
ments. These  filaments  may  ordinarily  be  removed  by  a  pipette 
since  they  are  generally  macroscopic. 

Tissue  Debris. — Masses  of  cells  or  fragments  of  tissue  are  fre- 
quently found  in  the  urinary  sediment.  They  may  be  found  in  the 
sediment  in  tubercular  affections  of  the  kidney  and  urinary  tract  or 


Fig.  116. — Human  Spermatozoa. 


in  tumors  of  these  organs.  Ordinarily  it  is  necessary  to  make  a  histo- 
logical examination  of  such  tissue  fragments  before  coming  to  a  final 
decision  as  to  their  origin. 

Animal  Parasites. — The  cysts,  booklets,  and  membrane  shreds 
of  echinococci  are  sometimes  found  in  the  urinary  sediments.  Other 
animal  organisms  which  are  more  rarely  met  with  in  the  urine  are 
embryos  of  the  Filaria  sanguinis  and  eggs  of  the  Distoma  hcBmatobium 
and  Ascarides.  Animal  parasites  in  general  occur  most  frequently 
in  the  urine  in  tropical  countries. 

Micro-organisms. — Bacteria  as  well  as  yeasts  and  moulds  are 
frequently  detected  in  the  urine.  Both  the  pathogenic  and  non- 
pathogenic forms  of  bacteria  may  occur.  The  non-pathogenic  forms 
most  frequently  observed  are  micrococcus  urea,  bacillus  urecB,  and 
staphylococcus  urece  liquefaciens.  Of  the  pathogenic  forms  many  have 
been  observed,  e.  g.,  Bacterium  Coli,  typhoid  bacillus,  tubercle  bacillus, 
gonococcus,  bacillus  pyocyaneus,  and  proteus  vulgaris.  Yeast  and  moulds 
are  most  frequently  met  with  in  diabetic  urine. 


356  PHYSIOLOGICAL    CHEMISTRY. 

Fibrin. — Following  hiematuria,  fibrin  clots  are  occasionally  ob- 
served in  the  urinary  sediment.  They  are  generally  of  a  semi-gelatin- 
ous consistency  and  of  a  very  light  color,  and  when  examined  under 
the  microscope  they  are  seen  to  be  composed  of  bundles  of  highly 
refractive  fibers  Avhich  run  parallel. 

Foreign  Substances  Due  to  Contamination.-  Such  foreign 
substances  as  fibers  of  silk,  linen,  or  wool;  starch  granules,  hair,  fat, 
and  sputum,  as  well  as  muscle  fibers,  vegetable  cells,  anci  food  par- 
ticles are  often  found  in  the  urine.  Care  should  be  taken  that  these 
foreign  substances  are  not  mistaken  for  any  of  the  true  sedimentary 
constituents  alreadv  mentioned. 


CHAPTER  XXI. 
URINE:   CALCULI. 

Urinary  calculi,  also  called  concretions,  or  concrements  are  solid 
masses  of  urinary  sediment  formed  in  some  part  of  the  urinary  tract. 
They  vary  in  shape  and  size  according  to  their  location,  the  smaller 
calculi,  termed  sand  or  gravel,  in  general  arising  from  the  kidney  or  the 
pelvic  portion  of  the  kidney,  vv^hereas  the  large  calculi  are  ordinarily 
formed  in  the  bladder.  There  are  two  general  classes  of  calculi  as 
regards  composition,  i.  e.,  simple  and  compound.  The  simple  form  is 
made  up  of  but  a  single  constituent,  whereas  the  compound  type  con- 
tains two  or  more  individual  constituents.  The  structural  plan  of 
most  calculi  consists  of  an  arrangement  of  concentric  rings  about  a 
central  nucleus,  the  number  of  rings  frequently  being  dependent  upon 
the  number  of  individual  constituents  which  enter  into  the  structure 
of  the  calculus.  In  case  two  or  more  calculi  unite  to  form  a  single 
calculus  the  resultant  body  will  obviously  contain  as  many  nuclei  as 
there  were  individual  calculi  concerned  in  its  construction.  Under 
certain  conditions  the  growth  of  a  calculus  will  be  principally  in  only 
one  direction,  thus  preventing  the  nucleus  from  maintaining  a  central 
location.  The  qualitative  composition  of  urinary  calculi  is  dependent, 
in  great  part,  upon  the  reaction  of  the  urine,  e.  g.,  if  the  reaction  of  the 
urine  is  acid  the  calculi  present  will  be  composed,  in  great  part  at  least, 
of  substances  that  are  capable  of  depositing  in  acid  urine. 

According  to  Ultzmann,  out  of  545  cases  of  urinary  calculus,  uric 
acid  and  urates  formed  the  nucleus  in  about  81  per  cent  of  the  cases; 
earthy  phosphates  in  about  9  per  cent;  calcium  oxalate  in  about  6  per 
cent;  cystine  in  something  over  i  per  cent,  while  in  about  3  per  cent 
of  the  cases  some  foreign  body  comprised  the  nucleus. 

In  the  chemical  examination  of^  urinary  calculi  the  most  valuable 
data  are  obtained  by  subjecting  each  of  the  concentric  layers  of  the 
calculus  to  a  separate  analysis.  Material  for  examination  may  be 
conveniently  obtained  by  sawing  the  calculus  carefully  through  the 
nucleus,  then  separating  the  various  layers  or  by  scraping  off  from 
each  layer  (without  separating  the  layers)  enough  powder  to  conduct 
the  examination  as  outlined  in  the  scheme  (see  page  359). 

357 


35o  PHYSIOLOGICAL    CHEMISTRY. 

Varieties  of  Calculus. 

Uric  Acid  and  Urate  Calculi. —  Uric  acid  and  urates  con- 
stitute the  nuclei  of  a  large  proportion  (8i  per  cent)  of  urinary  concre- 
tions. Such  stones  are  always  colored,  the  tint  varying  from  a  pale 
yellow  to  a  brownish-red.  The  surface  of  such  calculi  is  generally 
smooth  but  it  may  be  rough  and  uneven. 

Phosphatic  Calculi. — Ordinarily  these  concretions  consist  prin- 
cipally of  "triple  phosphate"  and  other  phosphates  of  the  alkaline 
earths,  with  very  frequent  admixtures  of  urates  and  oxalates.  The 
surface  of  such  calculi  is  generally  rough  but  may  occasionally  be 
rather  smooth.  The  calculi  are  somewhat  variable  in  color,  exhibiting 
gray,  white,  or  yellow  tints  under  different  conditions.  When  com- 
posed of  earthy  phosphates  the  calculi  are  characterized  by  their 
friability. 

Calcium  Oxalate  Calculi. — This  is  the  hardest  form  of  calculus 
to  deal  with,  and  is  rather  difficult  to  crush.  They  ordinarily  occur 
in  two  general  forms,  i.  e.,  the  small,  smooth  concretion  which  is 
characterized  as  the  hemp-seed  calculus  and  the  medium-sized  or 
large  stone  possessing  an  extremely  uneven  surface  which  is  generally 
classed  as  a  mulberry  calculus.  This  roughened  surface  of  the  latter 
form  of  calculus  is  due,  in  many  instances,  to  protruding  calcium 
oxalate  crystals  of  the  octahedral  type. 

Calcium  Carbonate  Calculi. — Calcium  carbonate  concretions 
are  quite  common  in  herbivorous  animals,  but  of  exceedingly  rare 
occurrence  in  man.  They  are  generally  small,  white,  or  grayish  calculi, 
spherical  in  form  and  possess  a  hard,  smooth  surface. 

Cystine  Calculi. — The  cystine  calculus  is  a  rare  variety  of  cal- 
culus. Ordinarily  they  occur  as  small,  smooth,  oval,  or  cylindrical 
concretions  which  are  white  or  yellow  in  color  and  of  a  rather  soft 
consistency. 

Xanthine  Calculi.  -  This  form  of  calculus  is  somewhat  more 
rare  than  the  cystine  type.  The  color  may  vary  from  white  to  brown- 
ish-yellow. Very  often  uric  acid  and  urates  are  associated  with  xan- 
thine in  this  type  of  calculus.  U])()n  nihljing  a  xanlhinc  talculus  it 
has  the  jjropcrly  of  assuming  a  wax-like  ap])earance. 

Urostealith  Calculi.  This  form  of  calculus  is  extremely  rare. 
Such  concretions  are  composed  principally  of  fat  and  fatty  acid. 
When  moist  they  are  soft  and  elastic,  but  when  dried  they  become 
brittle.     Urostealiths  are  generally  light  in  color. 

Fibrin  Calculi.— Fibrin  calculi  are  produced  in   the  process  of 


URINE. 


359 


On  Heating 

the  Powder  on  Platinum  Foil,  It 

Does  not  burn 

Does  burn 

The  powder  when  treated  with  HCl 

With 

flame 

Without  flame 

1 

m   O   p 

E-P  3 
hd  CO  S 

p 

p 

CL 

Does  not  effervesce 

o  " 

g,hrj 

5'  U 

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ft 
ft  r= 

ft  ^ 

l-t  - 

tn    p 

S-3 

tn    f' 

V-.    ft 

^    t" 

ft 

der  gives  the 
murexide  test 

The  gently-heated  powder  with  HCl 

ft-  p 
i^^fT 

U-cr 

HI   CW 

5:^ 

The    pow- 
der     when 

The  powder  when  moistened 
with  a  little  KOH 

3  S 
><  2 

ft  5 

*T3 

p 

ft  ft 
tn  cr 

cr  -1 
ft  ft 
R  a. 

treated     with 
KOH  gives 

A 
solt 

o 

o  ° 
o3 

O    i= 

2.  o 

o 
o 
3 

3 

d 

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P 

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O   3" 
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360  PHYSIOLOGICAL    CHEMISTRY. 

blood  coagulation  within  the  urinary  tract.  They  frequently  occur 
as  nuclei  of  other  forms  of  calculus.    They  are  rarely  found. 

Cholesterol  Calculi. — An  extremely  rare  form  of  calculus  some- 
what resembling  the  cystine  type. 

Indigo  Calculi. — Indigo  calculi  are  extremely  rare,  only  two 
cases  ha\ing  been  reported.  One  of  these  indigo  calculi  is  on  exhibi- 
tion in  the  museum  of  Jefferson  Medical  College  of  Philadelphia. 

The  scheme,  proposed  by  Heller  and  given  on  page  359,  will  be 
found  of  much  assistance  in  the  chemical  examination  of  urinary 
calculi. 


CHAPTER  XXII. 
URINE:   QUANTITATIVE   ANALYSIS. 

I.  Protein. 

1.  Scherer's  Coagulation  Method. — The  content  of  coagulahle 
protein  may  be  accurately  determined  as  follows:  Place  50  c.c.  of 
urine  in  a  small  beaker  and  raise  the  temperature  of  the  fluid  to  about 
40°  C.  upon  a  water-bath.  Add  dilute  acetic  acid,  drop  by  drop,  to 
the  warm  urine,  to  precipitate  the  protein  which  will  separate  in  a 
flocculent  form.  Care  should  be  taken  not  to  add  too  much  acid; 
ordinarily  less  than  twenty  drops  is  sufficient.  The  temperature  of 
the  water  in  the  water-bath  should  now  be  raised  to  the  boiling-point 
and  maintained  there  for  a  few  minutes  in  order  to  insure  the  complete 
coagulation  of  the  protein  present.  Now  filter  the  urine^  through  a 
previously  ivashed,  dried,  and  weighed  filter  paper,  wash  the  precipitated 
protein,  in  turn,  with  hot  water,  95  per  cent  alcohol,  and  with  ether, 
and  dry  the  paper  and  precipitate,  to  constant  weight,  in  an  air-bath 
at  110°  C.  Subtract  the  weight  of  the  filter  paper  from  the  combined 
weight  of  the  paper  and  precipitate  and  calculate  the  percentage  of 
protein  in  the  urine  specimen. 

Calculation. — To  determine  the  percentage  of  protein  present  in 
the  urine  under  examination,  multiply  the  weight  of  the  precipitate, 
expressed  in  grams,  by  2. 

2.  Esbach's  Method. — This  method  depends  upon  the  precipi- 
tation of  protein  by  Esbach's  reagent^  and  the  apparatus  used  in  the 
estimation  is  Esbach's  albuminometer  (Fig.  117,  p.  362).  In  making 
a  determination  fill  the  albuminometer  to  the  point  U  with  urine, 
then  introduce  the  reagent  until  the  point  R  is  reached.  Now  stopper 
the  tube,  invert  it  slowly  several  times  in  order  to  insure  the  thorough 
mixing  of  the  fluids,  and  stand  the  tube  aside  for  24  hours.  Creatinine, 
resin  acids,  etc.,  are   precipitated  in  this  method,  and  for  this   and 

^  If  it  is  desired  the  precipitate  may  be  filtered  off  on  an  unweighed  paper,  and  its 
nitrogen  content  determined  by  tiie  Kjeldahl  method  (see  p.  375).  In  order  to  arrive  at 
correct  figures  for  the  protein  content  it  is  then  simply  necessary  to  multiply  the  total 
nitrogen  content  by  6  .  25  (see  p.  407).  Correction  should  be  made  for  the  nitrogen  content 
of  the  filter  paper  used  unless  this  factor  is  negligible. 

-  Esbach's  reagent  is  prepared  by  dissolving  10  grams  of  picric  acid  and  20  grams  of 
citric  acid  in  i  liter  of  water. 

361 


362 


PHYSIOLOGICAL    CHEMISTRY. 


iVii 


other  reasons  it  is  not  as  accurate  as  the  coagulation  method.     It  is, 

however,  extensively  used  clinically. 

Calculation. — The    graduations    on    the    albuminometer    indicate 

grams  of  protein  per  liter  of  urine.     Thus,  if  the  protein  precipitate  is 

level  with  the  figure  3  of  the  graduated  scale,  this  denotes  that  the  urine 
examined  contains  3  grams  of  protein  to  the  liter. 
To  express  the  amount  of  protein  in  per  cent  simply 
move  the  decimal  point  one  place  to  the  left.  In  the 
case  under  consideration  the  urine  contains  0.3  per 
cent  protein. 

3.  Kwilecki's Modification  of  Esbach's  Method.^ 
— Add  10  drops  of  a  10  per  cent  solution  of  FeClg  to 
the  acid  urine  before  introducing  the  Esbach's  reagent. 
Warm  the  tube  and  contents  in  a  water-bath  at  72° 
C.  for  5-6  minutes  and  make  the  reading. 

II.  Dextrose. 

I.  Fehling's  Method. — Place  10  c.c.  of  the  urine 
under  examination  in  a  100  c.c.  volumetric  flask  and 
make  the  volume  up  to  100  c.c.  with  distilled  water. 
Thorolighly  mix  this  diluted  urine  by  pouring  it  into 
a  beaker  and  stirring  with  a  glass  rod,  then  transfer 
a  portion  of  it  to  a  burette  which  is  properly  sup- 
ported in  a  clamp. 

Now  place  10  c.c.  of  Fehling's  solution^  in  a 
small  beaker,  dilute  it  with  approximately  40  c.c.  of 
distilled  water,  heat  to  boiling,  and  observe  whether 
decomposition  of  the  Fehling's  solution  itself  has 
occurred  as  indicated  by  the  production  of  a  turbidity. 
If  such  turbidity  is  produced  the  Fehling's  solution 
is  unfit  for  use.  Clamp  the  burette  containing  the 
dilute  urine  immediately  over  the  beaker  and  carefully 
allow  from  0.5  to  i  c.c.  of  the  diluted  urine  to  How  into 
the  boiling  Fehling's  solution,  iking  the  solution  to  the  b()iling-])oint 
after  each  addition  of  urine  and  continue  running  in  the  urine  from 
the  burette,  0.5-1  c.c.  at  a  time,  as  indicated,  until  the  Fehling's  solu- 
tion is  completely  reduced,  i.  e.,  until  all  the  cupric  oxide  in  solution 
has  been  precipitated  as  cuprous  oxide.     This  point  will  be  indicated 

'  Kwilecki:     Milnch.  Med.  Work.,  LVI,  p.  1.^30. 

^  Directions  for  the  preparation  of  Fehling's  solution  are  given  in  a  note  at  the  liottom 
of  page  27. 


Fig.  117. — Es- 
bach's Albumin- 
O.METER.    (Ogden.) 


urine:  quantitative  analysis.  363 

by  the  absolute  disappearance  of  all  blue  color.  When  this  end-point  is 
reached  note  the  number  of  cubic  centimeters  of  diluted  urine  used 
in  the  process  and  calculate  the  percentage  of  dextrose  present,  in  the 
sample  of  urine  analyzed,  according  to  the  method  given  below. 

This  is  a  very  satisfactory  method,  the  main  objection  to  its  use 
being  the  uncertainty  attending  the  determination  of  the  end-reaction, 
i.  e.,  the  difficulty  with  which  the  exact  point  where  the  blue  color 
finally  disappears  is  noted-.  Several  means  of  accurately  fixing  this 
point  have  been  suggested,  but  they  are  practically  all  open  to  objection. 
As  good  a  ''check"  as  any,  perhaps,  is  to  filter  a  few  drops  of  the  solu- 
tion, through  a  double  paper,  after  the  blue  color  has  apparently  dis- 
appeared, acidify  the  filtrate  with  acetic  acid  and  add  potassium 
ferrocyanide.  If  the  copper  of  the  Fehling's  solution  has  been  com- 
pletely reduced,  there  will  be  no  color  reaction,  whereas  the  produc- 
tion of  a  brown  color  indicates  the  presence  of  unreduced  copper. 
Harrison  has  recently  suggested  the  following  procedure  to  determine 
the  exact  end-point:  To  about  i  c.c.  of  a  starch  iodide  solutionMn  a 
test-tube  add  2-3  drops  of  acetic  acid  and  introduce  into  the  acidified 
mixture  1-2  drops  of  the  solution  to  be  tested.  Unreduced  copper  will 
be  indicated  by  the  production  of  a  purplish-red  or  blue  color  due  to 
the  liberation  of  iodine. 

It  is  ordinarily  customary  to  make  at  least  three  determinations 
by  Fehling's  method  before  coming  to  a  final  conclusion  regarding 
the  sugar  content  of  the  urine  under  examination. 

Calculation. — Ten  c.c.  of  Fehling's  solution  is  completely  reduced 
by  0.05  gram  of  dextrose.^  If  y  represents  the  number  of  cubic  centi- 
meters of  undiluted  urine  (obtained  by  dividing  the  burette  reading  by 
10)  necessary  to  reduce  the  10  c.c.  of  Fehling's  solution,  we  have  the 
following  proportion : 

y  :o.o5  ::  100  :x  (percentage  of  dextrose). 

'2.  Benedict's  Method. — To   30  c.c.   of  Benedict's  solution^  in 

^  The  starch-iodide  solution  may  be  prepared  as  follows:  Mix  o.i  gram  of  starch 
with  cold  water  in  a  mortar  and  pour  the  suspended  starch  granules  into  75-100  c.c.  of 
boiling  water,  stirring  continuously.  Cool  the  starch  paste,  add  20-25  grams  of  potassium 
iodide  and  dilute  the  mixture  to  250  c.c.  This  solution  deteriorates  upon  standing,  and 
therefore  must  be  freshly  prepared  as  needed. 

^  The  values  for  certain  other  sugars  are  as  follows : 

Lactose    o . 0676  gram. 

Maltose o .  074    gram. 

Invert  sugar 0.0475  gram. 

^  Benedict's  solution  used  in  the  quantitative  determination  of  sugar  consists  of  three 

separate  solutions.     The  cupric  sulphate  solution  and  the  alkaline  tartrate  solutioft  are  the 

same  as  those  already  described  in  connection  with  Benedict's  qualitative  test,  see  p.  309. 

The  third  solution  is  made  up  as  follows: 

Potassium  ferro-thiocyanate  solution  =  1^  grams  of  potassium  ferrocyanide,  62.5  grams 


364  PHYSIOLOGICAL    CHEMISTRY. 

a  small  beaker  add  from  2.5  grams  to  5  grams  of  anhydrous  sodium 
carbonate^  and  heat  the  mixture  to  boiling  over  a  wire  gauze  until  the 
carbonate  has  been  brought  into  solution. 

Place  the  urine  under  examination  in  a  burette  and  run  it  into  the 
hot  Benedict  solution  rather  rapidly'  until  the  formation  of  a  heavy 
ihalk-u'hile  precipitate  is  noted  and  the  blue  color  of  the  solution 
lessens  perceptibly  in  its  intensity.  From  this  point  in  the  determina- 
tion from  2  to  10  drops^  of  the  urine  should  be  run  into  the  boiling 
Benedict  solution  at  one  time,  boiling  the  solution  vigorously  for 
about  15  seconds  after  each  addition.  Complete  reduction  of  the 
copper  is  indicated  here  as  in  Fehling's  original  method,  by  the  com- 
plete disappearance  of  all  blue  color.  The  end-point  here,  however,  is 
very  sharply  defined,  contrary  to  the  conditions  in  the  older  method. 

To  prexent  the  annoying  bumping  which  often  interferes  with  the 
titration,  a  medium-sized  piece  of  washed  absorbent  cotton'  may  be 
introduced  into  the  solution.  This  cotton  may  be  stirred  about  through 
the  solution  as  the  titration  proceeds  and  the  bumping  thus  eliminated. 

Calculation. — Thirty  cubic  centimeters  of  Benedict's  solution  is 
completely  reduced  by  0.073  gram  of  dextrose.  If  y  represents  the 
number  of  cubic  centimeters  of  urine  necessary  to  reduce  the  30  c.c. 
of  the  solution  we  have  the  following  proportion : 

y  :  0.073  ::  100  :.v  (percentage  of  dextrose). 

3.  Purdy's  Method.  Purdy's  solution''  is  a  modification  of 
Fehling's  solution  and  is  said  to  possess  greater  stability  than  the 

t)f  potassium  thiocyanate  and  50  grams  of  anhydrous  sodiinn  carhonalt'  dissolved  in  water 
anfl  made  up  to  500  c.c. 

These  three  solutions  sh(juld  he  preserved  separately  in  rubher-stoijpcred  bottles  and 
mi.xcd  in  equal  volumes  when  needed  for  use.     This  is  done  to  prevent  deterioration. 

'  The  amount  adrled  depends  upon  the  dilution  to  whic  h  the  solution  is  to  he  subjected 
in  tilrati<jn.  Kor  this  reason  the  maximum  amount  of  sodium  (arhonate  should  he  added 
when  titrating  urines  containing  a  very  low  percentage  of  sugar. 

^  Not  rajji'ily  enough,  however,  to  interfere  in  any  marked  degree  with  the  (ontinuous 
vigorous  boiling  of  the  solution. 

'  The  cxa<  t  amount  t<j  run  in  depends  ujKin  the  intensity  of  the  remaining  blue  color, 
as  well  as  upon  the  sugar  content  of  the  urine.  The  10  (Irojjs  should  he  added  at  one 
time  only  when  urines  ccmtaining  a  very  low  percentage  of  sugar  arc  under  cxamiiKUion. 

*  (jiass  wool  may  he  substituted  if  desired. 

■'  Purdy's  solution  has  the  lollowing  comijosition ; 

Cujjrii   sulphate    4-7.S2  grams. 

Potassium  hydroxide   2.5 . 5       grams. 

■Ammonia  (U.  S.  P.,  sp.  gr.  0.9) .SSOO       '^•^■• 

Glycerol ,SS  .0       c.c. 

iJi.stilled  water,  to  make  total  vcjlume  1  liter. 

In  preparing  the  dilution  bring  the  i  uprii  sul|>hatc  and  p  itassium  hydroxide  into 
solution  in  separate  vessels,  mix  the  two  solutions,  cool  the  mixture,  and  add  the  ammonia 
and  glycerol.  After  this  has  been  done  the  total  volume  should  be  made  uj)  lo  1  liter  with 
distilled  water. 

Thirty-five  <  uI/k  imtimeters  f)f  Purdy's  solution  is  exa<lly  reduced  by  o.o.'  gram  of 
dextrose. 


urine:  quantitative  analysis.  365 

latter.  One  of  the  most  satisfactory  points  about  the  method  as  sug- 
gested by  Purdy  is  the  ease  with  which  the  exact  end-reaction  may 
be  determined.  In  determining  the  percentage  of  dextrose  by  this 
method  proceed  as  follows:  Place  35  c.c.  of  Purdy 's  solution  in  a  200 
c.c.  Erlenmeyer  flask  and  dilute  the  fluid  with  approximately  two 
volumes  of  distilled  water.  Fit  a  cork,  provided  with  two  perforations, 
to  the  neck  of  the  flask  and  through  one  perforation  introduce  the  tip 
of  a  burette  and  through  the  second  perforation  introduce  a  tube 
bent  at  right  angles  in  such  a  manner  as  to  allow  the  steam  to  escape 
and  keep  the  fumes  of  ammonia  away  from  the  face  of  the  operator 
as  completely  as  possible.^  Now  bring  the  solution  to  the  boiling- 
point  and  add  the  urine,  drop  by  drop,  until  the  intensity  of  the  blue 
color  begins  to  diminish.  When  this  point  is  reached  add  the  urine 
somewhat  more  slowly  until  the  blue  color  is  entirely  dissipated  and 
an  absolutely  decolorized  solution  remains.  Take  the  burette  reading 
and  calculate  the  percentage  of  dextrose  in  the  urine  examined  accord- 
ing to  the  method  given  below. 

Care  should  be  taken  not  to  boil  the  solution  for  too  long  a  period, 
since,  under  these  conditions,  sufficient  ammonia  might  be  lost  to 
allow  the  cuprous  hydroxide  to  precipitate. 

Some  investigators  consider  it  to  be  advisable  to  dilute  the  urine 
before  applying  the  above  manipulation,  but  ordinarily  this  is  not 
necessary  unless  the  urine  has  a  high  content  of  dextrose  (5  per  cent 
or  over).  In  this  event  the  urine  may  be  diluted  with  2-3  volumes  of 
water  and  the  proper  correction  made  in  the  calculation. 

Calculation. — Thirty-five  c.c.  of  Purdy 's  solution  is  completely 
reduced  by  0.02  gram  of  dextrose.  If  y  represents  the  number  of  cubic 
centimeters  of  undiluted  urine  necessary  to  reduce  35  c.c.  of  Purdy's 
solution,  we  have  the  following  proportion: 

y  :  0.02  :  :  100  -.x  (percentage  of  dextrose). 

4.  Fermentation  Method. — This  method  consists  in  the  measure- 
ment of  the  volume  of  carbon  dioxide  evolved  when  the  dextrose  of  the 
urine  undergoes  fermentation  with  yeast.  None  of  the  various  methods 
whose  manipulation  is  based  upon  this  principle  is  absolutely  accurate. 
The  method  in  which  Einhorn's  saccharometer  (Fig.  2,  page  31)  is  the 
apparatus  employed  is  perhaps  as  satisfactory  as  any  for  clinical  pur- 

]  This  side  tube  may  also  be  equipped  with  a  simple  air-valve,  thus  insuring  the  ex- 
clusion of  air  and  thereby  contributing  to  the  accuracy  of  the  determination,  inasmuch  as 
the  cuprous  salts  would  be  reoxidized  upon  coming  in  contact  with  the  air.  If  one  is 
careful  to  maintain  the  solution  continuously  at  the  boiling-point  throughout  the  entire 
process,  however,  there  is  no  opportunity  for  air  to  enter  and  therefore  no  need  of  an 
air-valve. 


366  PHYSIOLOGICAL   CHEMISTRY. 

poses.  The  procedure  is  as  follows:  Place  about  15  c.c.  of  urine  in  a 
mortar,  add  about  i  gram  of  yeast  (1/16  of  the  ordinary  cake  of  com- 
pressed yeast)  and  carefully  crush  the  latter  by  means  of  a  pestle. 
Transfer  the  mixture  to  the  saccharometer,  being  careful  to  note  that 
the  graduated  tube  is  completely  filled  and  that  no  air  bubbles  gather 
at  the  top.  Allow  the  apparatus  to  stand  in  a  warm  place  (30°  C.) 
for  1 2  hours,  and  observe  the  percentage  of  dextrose  as  indicated  by 
the  graduated  scale  of  the  instrument.  Both  the  percentage  of  dex- 
trose and  the  number  of  cubic  centimeters  of  carbon  dioxide  are  indi- 
cated by  the  graduations  on  the  side  of  the  saccharometer  tube. 

5.  Polariscopic  Examination. — Before  subjecting  urine  to  a 
polariscopic  examination  the  slightly  acid  fluid  should  be  decolorized 
as  thoroughly  as  possible  by  the  addition  of  a  little  plumbic  acetate. 
The  urine  should  be  well  stirred  and  then  filtered  through  a  filter  paper 
which  has  not  been  previously  moistened.  In  this  way  a  perfectly  clear 
and  almost  colorless  liquid  is  obtained.  , 

In  determining  dextrose  by  means  of  the  polariscope  it  should  be 
borne  in  mind  that  this  carbohydrate  is  often  accompanied  by  other 
optically  active  substances,  such  as  proteins,  laevulose,  /3-oxybutyric 
acid,  and  conjugate  glycuronates  which  may  introduce  an  error  into 
the  polariscopic  reading;  the  method  is,  however,  sufficiently  accurate 
for  practical  purposes. 

For  directions  as  to  the  manipulation  of  the  polariscope  see 
page  31. 

III.  Uric  Acid. 

I.  Folin-Shaffer  Method.— Introduce  100  c.c*  of  urine  into  a 
beaker,  add  25  c.c.  of  the  Folin-Shaffcr  reagent^  and  allow  the  mixture 
to  stand, ^  without  further  stirring,  until  the  precipitate  has  settled  (5- 
10  minutes).  Filter,  transfer  100  c.c.  of  the  filtrate  to  a  200  c.c.  beaker 
or  Erienmeyer  flask,  add  5  c.c.  of  concentrated  ammonium  hydroxide 
and  allow  the  mixture  to  stand  for  24  hours.  Transfer  the  precipitated 
ammonium  urate  f|uantilatively  to  a  filter  paper,*  using  10  per  cent 
ammonium  sulphate  to  remove  the  final  traces  of  the  urate  from  the 
beaker.     Wash  the  precipitate  approximately  free  from  chlorides  by 

'  It  is  preferable  to  use  more  than  100  c.c.  of  uritic  if  the  fluid  has  a  specific  gravity 
less  than  i  .020. 

*  The  Folin-Shaffer  reagent  consists  of  500  grams  of  ammonium  suljihatc,  5  grams  of 
uranium  acetate  and  60  c.c.  of  10  per  cent  acetic  acid  in  650  c.c.  of  distilled  water. 

^  The  mixture  should  not  be  allowed  to  stand  for  too  long  a  time  at  this  point,  since 
uric  acid  may  be  lost  through  precipitation. 

*  The  Schleicher  an'l  .Schull  hardened  papers  or  the  Baker  and  Adamaon  washed,  ashless 
variety  are  very  satisfactory  for  this  purpose. 


urine:  quantitative  analysis.  367 

means  of  10  per  cent  ammonium  sulphate  solution/  remove  the  paper 
from  the  funnel,  open  it,  and  by  means  of  hot  water  rinse  the  precipitate 
back  into  the  beaker  in  which  the  urate  was  originally  precipitated. 
The  volume  of  fluid  at  this  point  should  be  about  100  c.c.  Cool  the 
solution  to  room  temperature,  add  15  c.c.  of  concentrated  sulphuric 
acid  and  titrate  at  once  with  N/20  potassium  permanganate,  K2Mn208, 
solution.  The  first  tinge  of  pink  color  which  extends  throughout  the 
fluid  after  the  addition  of  two  drops  of  the  permanganate  solution, 
while  stirring  with  a  glass  rod,  should  be  taken  as  the  end-reaction. 
Take  the  burette  reading  and  compute  the  percentage  of  uric  acid 
present  in  the  urine  under  examination. 

Calculation. — Each  cubic  centimeter  of  N/20  potassium  permanga- 
nate solution  is  equivalent  to  3.75  milligrams  (0.00375  gram)  of  uric 
acid.  The  100  c.c.  from  which  the  ammonium  urate  was  precipitated 
is  equivalent  to  only  four-fifths  of  the  100  c.c.  of  urine  originally  taken, 
therefore  we  must  take  five-fourths  of  the  burette  reading  in  order  to 
ascertain  the  number  of  cubic  centimeters  of  the  permanganate  solu- 
tion required  to  titrate  100  c.c.  of  the  original  urine  to  the  correct  end- 
point.  If  y  represents  the  number  of  cubic  centimeters  of  the  per- 
manganate solution  required,  we  may  make  the  following  calculation: 

y  X  0.00375  =  weight  of  uric  acid  in  100  c.c.  of  urine. 

Because  of  the  solubility  of  the  ammonium  urate  a  correction  of  3 
milligrams  should  be  added  to  the  final  result. 

Calculate  the  quantity  of  uric  acid  in  the  twenty-four-hour  urine 
specimen. 

2.  Heintz  Method. — This  is  a  very  simple  method  and  was  the 
first  one  in  general  use  for  the  quantitative  determination  of  uric  acid. 
It  is  believed  to  be  somewhat  less  accurate  than  the  method  just 
described.  The  procedure  is  as  follows :  Place  100  c.c.  of  filtered  urine 
in  a  beaker,  add  5  c.c.  of  concentrated  hydrochloric  acid,  stir  the  fluid 
thoroughly,  and  stand  it  away  in  a  cool  place  for  24  hours.  Filter  off 
the  uric  acid  crystals  upon  a  washed,  dried  and  weighed  filter  paper 
and  wash  them  with  cold  distilled  water,  a  few  cubic  centimeters  at  a 
time  until  the  chlorides  are  removed.  Now  wash,  in  turn,  with  alcohol 
and  with  ether  and  finally  dry  the  paper  and  crystals  to  constant 
weight  at  110°  C.  In  the  process  of  washing  the  uric  acid  free  from 
chlorides  an  error  is  introduced,  since  every  cubic  centimeter  of  water 
so  used  dissolves  0.00004  gram  of  uric  acid.     For  this  reason  a  correc- 

^  This  washing  may  be  conveniently  done  by  decantation  if  desired,  thus  retaining  the 
major  portion  of  the  precipitate  in  the  beaker  or  flask. 


368  PHYSIOLOGICAL    CHEMISTRY. 

tion  is  necessary.  It  has  been  suggested  that  the  pigment  of  the  crys- 
tals is  equivalent  in  weight  to  the  amount  of  uric  acid  dissolved  by  the 
first  30  c.c.  of  water,  and  this  factor  should  be  taken  into  acct)unt  in  the 
computation  of  the  percentage  of  uric  acid. 

Cakidatian. — Since  100  c.c.  of  urine  was  used  the  co/'rcc/fJ.  weight 
of  the  uric  acid  crystals,  in  grams,  will  express  the  percentage  of 
uric  acid  present. 

3.  Kriiger  and  Schmidt's  Method. — This  method  serves  for  the 
detection  of  both  uric  acid  and  the  purine  bases.  The  principle 
inxohed  is  the  precipitation  of  both  the  uric  acid  and  the  purine  bases 
in  combination  with  copper  oxide  and  the  subsec[uent  decomposition 
of  this  precipitate  by  means  of  sodium  sulphide.  The  uric  acid  is  then 
precipitated  by  means  of  hydrochloric  acid  and  the  purine  bases  are 
separated  from  the  filtrate  in  the  form  of  their  copper  or  silver  com- 
pounds. The  nitrogen  content  of  the  precipitates  of  uric  acid  and  pur- 
ine bases  is  then  determined  by  means  of  the  Kjeldahl  method  (see  p. 
375)  and  the  corresponding  values  for  uric  acid  and  purine  bases  calcu- 
lated. The  method  is  as  follows:  To  400  c.c.  of  albumin-free  urine^ 
in  a  liter  flask, ■  add  24  grams  of  sodium  acetate,  40  c.c.  of  a  solution 
of  sodium  bisulphite''  and  heat  the  mixture  to  Ijoiling.  Add  40-80 
c.c'  of  a  10  per  cent  solution  of  cupric  sulphate  and  maintain  the 
temperature  of  the  mixture  at  the  boiling-point  for  at  least  three  min- 
utes. Filter  off  the  flocculent  precipitate,  wash  it  with  hot  water  until 
the  wash  water  is  colorless,  and  return  the  washed  precipitate  to  the 
flask  by  puncturing  the  tip  of  the  filter  paper  and  washing  the  precipi- 
tate through  by  means  of  hot  water.  Add  water  until  the  volume  in 
the  flask  is  approximately  200  c.c,  heat  the  mixture  to  boiling,  and 
decompose  the  i)reci[ntale  of  copper  oxide  l)y  the  addition  of  30  c.c. 
of  sodium  sulphide  solution.''  After  decom])osilion  is  complete,  the 
mixture  should  be  acidified  with  acetic  acid  and  heated  to  boiling  until 
the  separating  sulphur  collects  in  a  mass.  Filter  the  hot  fluid  by  means 
<;f  a  filler  pump,  wash  with  hot  waUr,  add  10  ex.  of  10  per  cent  hydro- 

'  If  alljuniin  is  prL-sent,  tlio  urine  sIkhiM  \n-  licaicil  In  liniliii)^,  ;i(  idilicil  wiih  ,i(i-tic  ;ui(l 
and  filtered. 

*  The  total  volume  of  urine  for  llie  twcnly-'our  Imuis  should  lie  siiHn  icnlly  dilulcd 
with  water  to  make  the  total  volume  of  the  solution  1600-2000  i  .c  . 

'A  soluti(jn  (ontaininx  50  j^rams  r)f  Kaliihaum's  commcn  i.il  sodium  Msulphilc  in 
100  ex..  of  water. 

*  The  exact  amount  depending;  u|)on  Uie  t onlent  of  the  purine  bases. 

' 'I'his  is  made  f)y  saturating  a  1  jier  cent  solution  of  sodium  iiydroxide  wiili  liy<lro).;cn 
sulphide  gas  and  adding  an  e(jual  volume  of  1  per  cent,  sodium  liydro.\idc. 

Orflinarily  the  addition  of  ,^0  c.c.  of  this  solution  is  sulVuient,  hut  the  preseiK  e  of  ;in 
exces.s  of  suljihide  should  he  proven  by  adding  a  dro|)  of  lead  arclalc  lo  a  drop  of  the 
solution.  Under  the.se  conditions  a  dark  brown  1  olor  will  show  ihc  prcscnrc  of  an  excess 
of  sorlium  snl[)hide 


urine:  quantitative  analysis. 


569 


chloric  acid  and  evaporate  the  filtrate  in  a  porcelain  dish  until  the  total 
volume  has  been  reduced  to  about  ten  cubic  centimeters.  Permit  this 
residue  to  stand  about  two  hours  to  allow  for  the  separation  of  the  uric 
acid,  leaving  the  purine  bases  in  solution.  Filter  off  the  precipitate 
of  uric  acid,  using  a  small  filter  paper,  and  wash  the  uric  acid,  with 
water  made  acid  with  sulphuric  acid,  until  the  total  volume  of  the 
original  filtrate  and  the  wash  water  aggregates  75  c.c  Determine  the 
nitrogen  content  of  the  precipitate  by 
means  of  the  Kjeldahl  method  (see  p. 
375)  and  calculate  the  uric  acid  equiva- 
lent. 

Calculation. — In  calculating  the  uric 
acid  value  from  the  total  nitrogen  simply 
multiply  the  latter  by  three  and  add  0.0035 
to  the  product  as  a  correction  for  the  uric 
acid  remaining  in  solution  in  the  75  c.c. 

IV.    Urea. 

I.  Knop-Hiifner  Hypobromite 
Method  (using  Marshall's  Urea  Ap- 
paratus).— Place  the  thumb  over  the 
side  opening  of  the  bulbed-tube  of  the 
apparatus  (Fig.  118)  and  carefully  fill 
the  tube  with  sodium  hypobromite  solu- 
tion.^ Close  the  opening  in  the  end  of 
the  tube  with  a  rubber  stopper,  incline 
the  tube  to  allow  air-bubbles  to  escape, 
and  finally  invert  the  tube  and  fix  the   . 

stoppered  end  in  the  saucer-shaped  vessel.  By  means  of  the  graduated 
pipette  rapidly  introduce  i  c.c.  of  urine"  into  T;he  hypobromite  solution 
through  the  side  opening  of  the  bulbed-tube.  Withdraw  the  pipette 
immediately  after  the  urine  has  been  introduced.  When  the  de- 
composition of  the  urea  is  completed  (10-20  minutes)  gently 
tap  the    bulbed-tube    with    the    finger    in    order    to     dislodge    any 


■  i'li|lllllllllllillllllllll'IH'|l|lllllllll!illl'ili'ilMii;!l!--~: 

Fig.    118. — Marshall's    Urea 
Apparatus.     (Tyson.) 
a,    Bulbed   measuring  tube;    b, 
saucer-shaped  vessel;  c,  graduated 
pipette;  d,  funnel-tube. 


'  The  ingredients  of  the  sodium  hypobromite  solution  should  be  prepared  in  the  form 
of  two  separate  solutions.  When  needed  for  use  mix  one  volume  of  solution  a,  one  volume 
of  solution  b,  and  3  volumes  of  water. 

(a)  Dissolve  125  grams  of  sodium  bromide  in  water,  add  125  grams  of  bromine  and 
make  the  total  volume  of  the  solution  i  liter. 

(b)  A  solution  of  sodium  hydroxide  having  a  specific  gravity  of  i .  250.  This  is  approxi- 
mately a  22.5  per  cent  solution. 

Preserve  both  solutions  in  rubber-stoppered  bottles. 

-  Ordinarily  i  c.c.  of  urine  is  sufficient;  more  may  be  used,  however,  if  its  content  of 
urea  is  very  low. 

24 


37©  PHYSIOLOGICAL    CHEMISTRY, 

gas  bubbles  which  may  have  collected  on  the  inner  surface  of 
the  glass.  The  atmospheric  pressure  should  now  be  equalized  by 
attaching  the  funnel-tube  to  the  bulbed-tube  at  the  side  opening  and 
introducing  hypobromite  solution  into  it  until  the  columns  of  liquid  in 
the  two  tubes  are  uniform  in  height.  The  graduated  scale  of  the 
bulbed-tube  should  now  be  read  in  order  to  determine  the  number  of 
cubic  centimeters  of  nitrogen  gas  evolved.  By  means  of  the  appended 
formula  the  n'eight  of  the  urea  present  in  the  urine  under  examination 
may  be  computed. 

Calculation} — By  properly  substituting  in  the  following  formula 
the  "weight  of  urea,  in  grams,  contained  in  the  volume  of  urine  decom- 
posed (i  c.c.  or  more)  may  readily  be  determined: 

3  54- 5X760(1-1-0. 003  665/) 
w;  =  weight  of  urea,  in  grams. 

'y  =  observed  volume  of  nitrogen  expressed  in  cubic  centimeters. 
/>  =  barometric  pressure  expressed  in  mm.  of  mercury. 
T===  tension  of  aqueous  vapor^  for  temperature  /. 
/  =  temperature  (centigrade). 

If  we  wish  to  calculate  the  percentage  of  urea  we  may  do  so  by 
means  of  the  following  proportion  in  which  y  represents  the  volume 
of  urine  used  and  w  denotes  the  weight  of  the  urea  contained  in  the 
volume  y. 

y  '.w  ::  \oo  :.v  (percentage  of  urea). 

Sodium  hypobromite  solution  may  also  be  employed  for  the  deter- 
mination of  urea  in  the  apparatus  devised  by  Hiifncr  which  is  pictured 
in  Fig.  119,  page  371. 

2.  Knop-Hiifner  Hypobromite  Method  (Using  the  Doremus- 
Hinds  Ureometer). — In  common  with  the  method  already  described, 
this  method  depends  upon  the  measurement  of  the  volume  of  nitrogen 
gas  liberated  when  the  urea  of  the  urine  is  decomj)oscd  by  means  of 
sodium  hypobromite  solulion.     'I'lic  Dorcmus-Ilinds  ureometer  (Fig. 

'  0.003665  =  coefficient  of  expansion  of  gases  for  1°  C.     354.5  =  11111111)01-  of  c.c.  of 

nitrogen  gas  evolved  from  i  gram  of  urea. 

^  The  values  of  T  for  the  temperatures  onliiiHiily  mil  wiili  ;uc  f^ivcn  in  Ihr  following 

table: 

Temp.                        Tension  in  mm.  Temp.                         Tension  in  mm. 

15°  C 12.677  21°  C 18.505 

16°  C 1.3-519  22°  C 19-675 

17°  C 14.009  23°  C 20.909 

18°  C 15-351  24°  C 22.211 

19°  C 16.345  25°  C 23.582 

20°  C 17  .396 


urine:  quantitative  analysis. 


371 


120,  p.  372),  is  one  of  the  simplest  and  cheapest  forms  of  apparatus  in 
general  use  for  the  determination  of  urea  by  the  hypobromite  process. 
In  using  this  apparatus  proceed  as  follows:  Fill  the  side  tube  B  and 
the  lumen  of  the  stopcock  C  with  the  urine  under  examination.  Care- 
fully wash  out  tube  A  with  water  and  introduce  into  it  sodium  hypo- 
bromite solution/  being  careful  to  fill 
the  bulb  sufficiently  full  to  prevent  the 
entrance  of  air  into  the  graduated 
portion.  Now  allow  i  c.c.  of  urine^ 
to  flow  from  tube  B  into  tube  A  and 
after  the  evolution  of  gas  bubbles  has 
ceased  (10-20  minutes)  take  the  read- 
ing of  the  graduated  scale  on  tube  A. 

In  common  with  all  other  methods 
which  are  based  upon  the  decomposi- 
tion of  urea  by  means  of  hypobromite 
solution,  this  method  is  not  absolutely 
correct.  It  is,  however,  sufficiently 
accurate  for  ordinary  clinical  purposes. 

Calculation. — Observe  the  reading 
on  the  'graduated  scale  of  tube  A. 
This  tube  is  so  graduated  as  to 
represent  the  weight  of  urea,  in  grams, 
per  cubic  centimeter  of  urine.  If  we 
wish  to  compute  the  percentage  of  urea 
present  this  may  be  done  very  readily 
by  simply  moving  the  decimal  point 
two  places  to  the  right;  e.  g.,  if  the  read- 
ing is  0.02  gram  the  urine  contains  2 
per  cent  of  urea. 

3.  Folin's  Method. — This  is  one 
of  the  most  accurate  methods  yet 
devised  for  the  determination  of  urea 
in  the  urine.  The  procedure  is  as  follows:  Place  5  c.c.  of  urine 
in  a  200  c.c.  Erlenmeyer  flask  and  add  to  it  5  c.c.  of  concentrated 
hydrochloric  acid,  20  grams  of  crystallized  magnesium  chloride,  a 
piece  of  paraffin  the  size  of  a  hazel  nut,  and  2-3  drops  of  a  i  per 
cent    aqueous    solution    of    "alizarin    red."      Insert    a    Folin   safety 

*  For  directions  as  to  the  preparation  of  this  solution  see  page  369. 

"  If  the  content  of  urea  in  the  urine  under  examination  is  large,  the  urine  may  be  diluted 
with  water  before  determining  the  urea.  If  this  is  done  it  must  of  course  be  taken  into 
consideration  in  computing  the  content  of  urea. 


Fig.    119. — Hufner's   Urea 
Apparatus. 


o/-' 


PHYSIOLOGICAL    CHEMISTRY. 


tube  (Fig  121,  p.  373)  into  the  neck  of  the  tiask  and  boil  the  mixture 
until  each  drop  of  retiow  from  the  safety  tube  produces  a  very 
perceptible  bump;  the  heat  is  then  reduced  somewhat  and  continued 
one  and  one-half  hours.  The  contents  of  the  flask  must  not  remain 
alkaline,  and  to  obviate  this,  at  the  first  appearance  of  a  reddish  tinge 
in  the  contents  of  the  flask  a  feu'  drops  of  the  acid  distillate  are  shaken 
f"^  back  into  the  flask.     Al  the  end  of  i  1/2 

hours  the  contents  of  the  \essel  are 
transferred  to  a  i-liter  flask  with  about 
700  c.c.  of  distilled  water,  about  20 
c.c.  of  10  per  cent  potassium  hydroxide 
or  sodium  hydroxide  solution  is  added 
and  the  mixture  distilled  into  a  known 
volume  of  N/io  sulphuric  acid  until  the 
contents  of  the  flask  are  nearly  dry  or 
until  the  distillate  fails  to  give  an 
alkaline  reaction  to  litmus,  showing  the 
absence  of  ammonia.  The  time  devoted 
to  this  process  is  ordinarily  about  an 
hour.  Boil  the  distillate  a  few  moments 
to  free  it  from  CO,,  then  cool  and 
titrate  the  mixture  with  N/io  sodium 
hydroxide,  using  "alizarin  red"  as  in- 
dicator. 

A  "check"  experiment  should 
always  be  made  to  determine  the 
original  ammonia  content  of  the  urine 
and  of  the  magnesium  chloride,  if  it  is 
nol  absolutely  ])ure,  which  of  course 
should  be  subtracted  from  the  tolal  amounl  of  ammonia  as  determined 
by  the  above  j)rocess. 

The  Folin  method  is  extremely  accurate  undiT  all  conditions  ixcept 
when  the  urine  contains  sugar.  When  this  is  the  case  the  carbohydrate 
and  the  urea  unite,  upon  being  healed,  and  form  a  very  stable  tombi 
nation.  P'or  this  reason  the  P'olin  method  is  not  suitable  for  use  in  the 
examinati(;n  of  su(  h  urines.  The  best  method  for  use  under  such 
condilons  is  the  (ombination  .M()rncr  SJoqvist  I'olin  method  which  is 
gi\en  below. 

4.  Morner-Sjogvist-Folin  Method.  As  has  already  been  slated 
in  the  last  exjjerimenl,  this  method  excels  the  Folin  method  in  accuracy 
only  in  the  determination  of  urea  in  the  presence  of  carbohydrate  bodies. 


Fig.  120. — UoRK.\ius-HiNDs  Ure- 

OMKTER. 


urine:  quantitative  analysis. 


;7: 


Briefly,  the  procedure  is  as  follows:^  Bring  the  major  portion  of  1.5 
gram  of  powdered  barium  hydroxide  into  solution  in  5  c.c.  of  urine  in 
a  small  flask,  and  treat  the  mixture  with  100  c.c.  of  an  alcohol-ether 
solution,  consisting  of  two  volumes  of  97  per  cent  alcohol  and  one  vol- 
ume of  ether.  Stopper  the  flask  and  allow  it  to  stand  12-24  hours. 
Filter  off  the  precipitate,  wash  it  with  the  alcohol-ether  mixture  and 
remove  the  alcohol  and  ether  from 
the  filtrate  by  distillation,  being  care- 
ful to  keep  the  temperature  of  the 
mixture  below  50°  C.^  Treat  the  re- 
maining fluid  (about  25  c.c.)  with  2 
c.c.  of  hydrochloric  acid  (sp.  gr.  1.124), 
transfer  it  carefully  to  a  200  c.c.  flask, 
and  evaporate  the  mixture  to  dryness 
on  a  water-bath.  Now  add  20  grams 
of  crystalHzed  magnesium  chloride 
and  2  c.c.  of  concentrated  hydro- 
chloric acid  to  the  residue,  and  after 
fitting  the  flask  with  a  return  cooler 
boil  the  mixture  on  a  wire  gauze  over 
a  small  flame  for  two  hours.  Cool  the 
solution,  dilute  to  750  c.c.  or  1000  c.c. 
with  water,  render  the  mixture  alka- 
line with  potassium  hydroxide  or 
sodium  hydroxide,  distil  off  the  am- 
monia and  collect  it  in  an  acid  solu- 
tion of  known  strength.  Boil  the 
distillate  to  remove  carbon  dioxide, 
cool  and  titrate  with  an  alkali  of 
known  strength.  In  this  method,  as 
well  as  in  Folin's  method  (see  p.  371),  correction  must  be  made  for 
the  ammonia  originally  present  in  the  urine  and  in  the  magnesium 
chloride. 

5.  Benedict's  Method.^ — Five  cubic  centimeters  of  urine  are  intro- 
duced into  a  rather  wide  test-tube,  about  3  grams  of  potassium  bisulphate 
and  1-2  grams  of  zinc  sulphate  added,  a  small  quantity  of  powdered 
pumice  and  a  bit  of  paraffin  are  introduced  and  the  mixture  boiled 
almost  to  dryness  either  over  a  free  flame  or  by  immersion  in  a  sul- 

'  The  original  description  of  the  method  may  be  found  in  an  article  by  Morner:  Skaii- 
dinavisches  Archiv/iir  Physiologic,  1903,  XIV,  p.  297. 
-  There  is  some  decomposition  of  urea  at  60°  C. 
'  Private  communication  from  Dr.  S.  R.  Benedict. 


Fig. 


-Folin's  Urea  App.ar.a.tus. 


•4 


PHYSIOLOGICAL   CHEMISTRY. 


phuric  acid  bath  at  about  130°.  The  tubes  are  then  weighted  (a  screw 
clamp  is  convenient)  and  immersed  for  three-fourths  of  their  length 
in  a  bath  of  sulphuric  acid  at  a  temperature  of  160-163°  ^^r  one  hour. 
The  residue  in  the  tube  is  then  dissolved  in  water  and  distilled  as  usual 
(see  Kjeldahl  Method,  p.  375),  boiling  with  sodium  carbonate  in  place 
of  hydroxide. 

V.  Ammonia. 

I.  Folin's  Method. — Place  25  c.c.  of  urine  in  an  aerometer  cylinder, 
30-40  cm.  in  height  (Fig.  122,  below),  add  about  i  gram  of  dry  sodium 
carbonate  and  introduce  some  crude  petroleum  to  prevent  foaming. 


Fig.  122. — Folin's  Ammonia  Apparatus. 


Insert  into  the  neck  of  the  cylinder  a  rubber  stopper  provided  with  two 
perforations,  into  each  of  which  passes  a  glass  tube,  one  of  which  reaches 
below  the  surface  of  the  liquid.  The  shorter  tube  (jo  cm.  in  length) 
is  connected  with  a  calcium  chloride  tube  filled  with  cotton,  and  this 
tube  is  in  turn  joined  to  a  glass  tube  extending  to  the  bottom  of  a  500 
c.c.  wide-mouthed  flask  which  is  intended  to  absorb  the  ammonia 
and  for  this  purpose  should  contain  20  c.c.  of  N/io  sulphuric  acid,  200 
c.c.  of  distilled  water  and  a  few  drops  of  an  indicator  ("alizarin  red"). 
To  insure  the  complete  aljsorption  of  the  ammonia  the  absorjjtion  flask 
is  provided  wilh  a  Folin  improved  absorption  tube  (l''ig.  123,  j).  375) 
which  is  very  effective  in  causing  the  air  passing  from  the  cylinder  to 
come  into  intimate  contact  with  the  acid  in  the  absorption  flask.  In 
order  to  exclude  any  error  due  to  the  presence  of  ammonia  in  the  air  a 


urine:  quantitative  analysis. 


375 


similar  absorption  apparatus  to  the  one  just  described  is  attached  to 
the  other  side  of  the  aerometer  cylinder,  thus  insuring  the  passage  of 
ammonia-free  air  into  the  cylinder.  With  an  ordi- 
nary filter  pump  and  good  water  pressure  the 
last  trace  of  ammonia  should  be  removed  from 
the  cylinder  in  about  one  and  one-half,  hours. ^ 
The  number  of  cubic  centimeters  of  the  N/io  sul- 
phuric acid  neutralized  by  the  ammonia  of  the 
urine  may  be  determined  by  direct  titration  with 
N/io  sodium  hydroxide. 

This  is  one  of  the  most  satisfactory  methods 
yet  devised  for  the  determination  of  ammonia. 
Steele^  has  recently  suggested  a    modification. 

Calculation. — Subtract  the  number  of  cubic 
centimeters  of  N/io  sodium  hydroxide  used  in  the 
titration  from  the  number  of  cubic  centimeters 
of  N/io  sulphuric  acid  taken.  The  remainder  is 
the  number  of  cubic  centimeters  of  N/io  sulphuric 
acid  neutralized  by  the  NH^  of  the  urine,  i  c.c.  of 
N/io  sulphuric  acid  is  equivalent  to  0.0017  S^ci'^ 
of  NH^.  Therefore  if  y  represents  the  volume  of 
urine  used  in  the  determination  and  y'  the  number  of  cubic  centi- 
meters of  N/io  sulphuric  acid  neutralized  by  the  NH^  of  the  urine, 
we  have  the  following  proportion: 

y  :  100  ::y'  X0.0017  :x  (percentage  of  NHg  in  the  urine  examined). 

Calculate    the  quantity   of   NHg    in    the    twenty-four-hour    urine 
specimen. 


Fig.  123. — FoLiN 
Improved  Absoiip- 
TiON  Tube. 


VI.  Nitrogen. 

Kjeldahl  Method.^ — The  principle  of  this  method  is  the  con- 
version of  the  various  nitrogenous  bodies  of  the  urine  into  -ammo- 
nium sulphate  by  boiling  with  concentrated  sulphuric  acid,  the  subse- 
quent decomposition  of  the  ammonium  sulphate  by  means  of  a  fixed 
alkali  (NaOH)  and  the  collection  of  the  liberated  ammonia  in  an 
acid  of  known  strength.     Finally,  this  partly  neutralized  acid  solu- 

^  With  any  given  filter  pump  a  "check"  test  should  be  made  with  urine  or  better  with  a 
solution  of  an  ammonium  salt  of  known  strength  to  determine  how  long  the  air  current 
must  be  maintained  to  remove  all  the  ammonia  from  25  c.c.  of  the  solution. 

^Steele:     Proc.  Soc.  Exp.  Biol,  and  Med.,  6,  p.  127. 

*  There  are  numerous  modifications  of  the  original  Kjeldahl  method ;  the  one  described 
here,  however,  has  given  excellent  satisfaction  and  is  recommended  for  the  determination 
of  the  nitrogen  content  of  urine. 


376  PHYSIOLOGICAL    CHEMISTRY. 

lion  is  titrated  with  an  alkali  of  known  strength  and  the  nitrogen  con- 
tent of  the  urine  under  examination  computed. 

The  procedure  is  as  follows:  Place  5  c.c.  of  urine  in  a  500  c.c. 
long-necked  Jena  glass  Kjeldahl  flask,  add  20  c.c.  of  concentrated 
sulphuric  acid  and  about  0.2  gram  of  cupric  sulphate  and  boil  the 
mixture  for  some  time  after  it  is  colorless  (about  one  hour.)  Allow 
the  flask  to  cool  and  dilute  the  contents  with  about  200  c.c.  of  ammonia- 
free  water.  Add  a  little  more  of  a  concentrated  solution  of  NaOH  than 
is  necessary  to  neutralize  the  sulphuric  acid^  and  introduce  into  the 
flask  a  little  coarse  pumice  stone  or  a  few  pieces  of  granulated  zinc,^  to 
prevent  bumping,  and  a  small  piece  of  paraffin  to  lessen  the  tendency 
to  froth.  By  means  of  a  safety-tube  connect  the  flask  with  a  con- 
denser so  arranged  that  the  delivery-tube  passes  into  a  vessel  con- 
taining a  known  volume  (the  volume  used  depending  upon  the  nitro- 
gen content  of  the  urine)  of  N/io  sulphuric  acid,  using  care  that  the 
end  of  the  delivery-tube  reaches  beneath  the  surface  of  the  fluid."' 
Mix  the  contents  of  the  distillation  flask  \-ery  thoroughly  by  shaking 
and  distil  the  mixture  until  its  volume  has  diminished  about  one-half. 
Titrate  the  partly  neutralized  N/ 10  sulphuric  acid  solution  by  means  of 
N  10  sodium  hydroxide,  using  congo  red  as  indicator,  and  calculate 
the  content  of  nitrogen  of  the  urine  examined. 

Calculation. — Subtract  the  number  of  cubic  centimeters  of  N/io 
sodium  hydroxide  used  in  the  titration  from  the  number  of  cubic 
centimeters  of  N/io  sulphuric  acid  taken.  The  remainder  is  equiva- 
lent to  the  number  of  cubic  centimeters  of  N/io  sulphuric  acid,  nen- 
tralized  by  the  ammonia  of  the  urine.  One  c.c.  of  N/10  sulphuric  acid 
is  equivalent  to  0.0014  gra^n  0/  nitrogen.  Therefore,  if  y  represents 
the  volume  of  urine  used  in  the  determination,  and  y'  the  number  of 
cubic  centimeters  of  N/io  sulphuric  a.c\d  neutralized  hy  the  ammonia  of 
the  urine,  we  ha\e  the  following  proportion: 
y  :  100  :  :  y'  X  0.0014  :  .v  (percentage  of  nitrogen  in  ihe  urine  examined). 

Calculate  the  (|uantity  of  nitrogen  in  the  twenty  four  hour  urine 
specimen. 

VII.  Hippuric  Acid. 

Dakin's  Methods.'' — Preliminary  Proerdure. — Place  1  50  c.c.  (or 
nKjrej    oi    the    urine    under   examination    in    a    porcelain    e\a|)ora(ing 

'  This  (oncenlratcd  sodiuni  liydroxi'lc-  soliiliDii  sliould  Ik-  |)iT|)iirr<i  in  (|ii;intity  and 
"check"  tests  made  U>  determine  tlie  voiumi-  of  Uie  solution  necessary  l<»  neiilraii/.e  tiic 
volume  (20  ( .1 .)  of  concentrated  sul|)hiiri<   .u  irl  used. 

^  Powdered  /.in(    may  lie  suhstiluted. 

'This  fleiivery-tube  should  he  of  lar^i-  (aiilici  in  oidcr  lo  avoid  llir  "  sui  I  inj^  liai  I;" 
of  the  fluid. 

'  Private  communication  to  ilic  aullior  from  l)i.  II.  I>.  l)akin. 


urine:  quantitative  analysis.  '  377 

dish  and  evaporate  almost  to  dryness  upon  a  water-bath.  Add  about 
I  gram  of  sodium  dihydrogen  phosphate,  about  25  grams  of  gypsum 
(CaSO^,  2H2O)  and  rub  up  with  a  pestle  and  stir  with  a  spatula  until 
a  uniform  mixture  results.  Dry  the  powder  thus  produced  in  a  water- 
oven  for  about  two  hours,  at  the  end  of  which  period  it  should  be  rubbed 
up  a  second  time,  to  remove  lumps,  and  transferred  to  a  Schleicher 
and  Schiill  "extraction  shell"  and  extracted  in  a  Soxhlet  apparatus 
in  the  usual  way  (see  p.  405).  The  extraction  medium  is  ethyl  acetate 
and  the  flask  containing  the  acetate  should  be  strongly  heated  over  a 
sand-hath^  for  about  two  hours.  The  ethyl  acetate  extract  is  now 
transferred  to  a  separatory  funnel,  and  the  original  flask  rinsed  with 
sufflcient  fresh  ethyl  acetate  to  make  the  total  volume  in  the  separatory 
funneP  about  100  c.c.  Wash  the  ethyl  acetate  solution  yzi;g  times  with 
a  saturated  solution  of  sodium  chloride,  using  8  c.c.  of  the  sodium 
chloride  solution  at  each  extraction,  shaking  vigorously  and  removing 
the  sodium  chloride  extract  in  each  case  before  adding  fresh  sodium 
chloride  solution.  The  sodium  chloride  removes  the  urea  completely 
and  the  hippuric  acid  is  then  determined  in  the  urea-free  solution  by 
the  following  volumetric  or  gravimetric  procedure: 

1.  Volumetric  Determination. — Transfer  the  urea-free  ethyl  acetate 
solution,  prepared  as  described  above,  to  a  Kjeldahl  flask,  add  about 
25  c.c.  of  water,  a  small  piece  of  pumice  stone,  to  prevent  bumping, 
attach  a  condenser  and  distil  off  the  ethyl  acetate^  over  a  free  flame. 
After  practically  all  of  the  ethyl  acetate  has  been  distilled  off,  the  nitro- 
gen in  the  remaining  solution  should  be  determined  by  means  of  the 
Kjeldahl  method  (see  p.  375). 

The  main  source  of  error  in  this  method  is  the  fact  that  any  nitro- 
gen present  in  the  form  of  phenaceturic  acid  or  indole  acetic  acid  is 
determined  as  hippuric  acid  nitrogen.  The  error  from  this  source 
is,  however,  usually  trifling. 

Calcidation. — Calculate  as  usual  for  nitrogen  determinations,  re- 
membering that  I  c.c.  o/N/io  sidphuric  acid  is  equivalent  to  0.0179  gf'om 
hippuric  acid. 

2.  Gravimetric  Determination. — The  urea-free  ethyl  acetate  so- 
lution, contained  in  the  separatory  funnel,  after  washing  with  sodium 
chloride  solution,  as  described  under  Preliminary  Procedure,  p.  376, 
is  washed  with  5  c.c.  of  distilled  water  to  remove  the  major  portion  of 

'  A  water-bath  cannot  be  substituted  inasmuch  as  the  resultant  extraction  would  be  too 
slow. 

-  This  ethyl  acetate  solution  contains  hippuric  acid,  urea,  and  other  substances. 

^  The  ethyl  acetate  after  separation  from  the  watery  layer  of  the  distillate  may  be  dried 
over  calcium  chloride  and  used  again. 


378  PHYSIOLOGICAL    CHEMISTRY. 

the  sodium  chloride.  Transfer  the  solution  from  the  separator}" 
funnel  to  a  round-bottomed  flask  and  subject  it  to  a  steam  distillation 
in  the  usual  way.  A  slow  current  of  steam  should  be  used  while  the 
ethyl  acetate  is  being  distilled  off  and  later  a  more  rapid  current  may 
be  employed.  The  distillation  should  be  continued  for  twenty  minutes. 
Now  add  about  o.i  gram  of  charcoal  to  the  aqueous  solution  which 
is  heated  to  boiling  and  filtered  hot.  Evaporate  the  solution  in  a 
iveighed  Jena  glass  dish  on  a  water-bath  until  the  volume  of  the  solu- 
tion is  reduced  to  about  3  c.c.  Stand  the  dish  in  a  warm  place  until 
evaporation  is  complete  and  a  crystalline  residue  remains.  Wash  the 
residue,  in  turn,  with  2  c.c.  of  dry  ether,  and  i  c.c.  of  water,  dry  it  in 
an  air-bath  at  100°  C.  and  weigh.  If  it  is  so  desired  the  residue  may 
be  recrystallized  from  a  little  hot  water  and  the  melting-point  deter- 
mined. Pure  hippuric  acid  melts  at  187°  C.  Contamination  with 
phenaceturic  acid  may  be  detected  both  by  the  melting-point  and  the 
microscopical  characteristics. 

VIII.  Sulphur. 

I.  Total  Sulphates. — Folhi's  Method. — Place  25  c.c.  of  urine 
in  a  200-250  c.c.  Erlenmeyer  flask,  add  20  c.c.  of  dilute  hydrochloric 
acid^  (i  volume  of  concentrated  HCl  to  4  volumes  of  water)  and  gently 
boil  the  mixture  for  20-30  minutes.  To  minimize  the  loss  of  water 
by  evaporation  the  mouth  of  the  flask  should  be  covered  with  a  small 
watch  glass  during  the  boiling  process.  Cool  the  flask  for  2-3  minutes 
in  running  water,  and  dilute  the  contents  to  about  150  c.c.  by  means 
of  cold  water.  Add  10  c.c.  of  a  5  per  cent  solution  of  barium  chloride 
slowly,  drop  by  drop,  to  the  cold  solution.^  The  contents  of  the  flask 
should  not  be  stirred  or  shaken  during  the  addition  of  the  barium  chloride. 
Allow  the  mixture  to  stand  at  least  one  hour,  then  shake  up  the  solu- 
tion and  filter  it  through  a  weighed  Gooch  crucible.' 

Wash  the  precipitate  of  BaSO^  with  about  250  c.c.  of  cold  water, 
dry  it  in  an  air-bath  or  over  a  very  low  llamc,  then  ignite,''  cool  and 
weigh. 

'  If  it  is  desired,  50  c.c.  of  urine  and  4  c.c.  of  concentrated  acid  may  he  used  instead. 

^  A  dropper  or  capillary  funnel  made  from  an  orriinary  (;alcium  ciiloridc  tuhe  and  so 
con.structed  as  to  deliver  10  c.c.  in  2-3  minutes  is  recommended  for  use  in  adding  the 
barium  chloride. 

^  If  a  (ifKjch  crucible  is  not  available,  the  precipitate  of  BaSO^  may  be  filtered  oflf  upon  a 
washed  filter  paper  (Schleicher  &  Schull's,  No.  589,  blue  ribbon),  and  after  washing  the 
precipitate  with  about  250  c.c.  of  cold  water  the  paper  and  preci[)ilate  may  be  dried  in 
an  air-bath  or  over  a  low  flame.  The  ignition  may  then  be  carried  out  in  the  usual 
way  in  the  ordinary  platinum  or  porcelain  crucible.  In  this  case  correction  must  be  made 
for  the  weight  of  the  ash  of  the  filter  paper  used. 

*  Care  must  be  taken  in  the  ignilif>n  of  predpilatcs  in  Gooch  crucibles.  The  flame 
should  never  be  applied  directly  to  the  perforated  bottom  or  to  the  sides  of  the  crucible, 


urine:  quantitative  analysis.  379 

Calculation. — Subtract  the  weight  of  the  Gooch  crucible  from 
the  weight  of  the  crucible  and  the  BaSO^  precipitate  to  obtain  the 
weight  of  the  precipitate.  The  weight  of  SOj^  in  the  volume  of  urine 
taken  may  be  determined  by  means  of  the  following  proportion. 

Mol.  wt.        Wt.  of       Mol.  wt. 

BaSO^rBaSO^:  iSOgirx;  (wt.  of  SO3  in  grams), 
ppt. 

Representing  the  weight  of  the  BaSO^  precipitate  by  y  and  substi- 
tuting the  proper  molecular  weights,  we  have  the  following  proportion: 

231.7:  y  :   :  79.5  :x  (wt.  of  SO3  in  grams  in  the  quantity  of  urine  used). 

Calculate  the  quantity  of  SO3  in  the  twenty-four-hour  specimen 
of  urine. 

To  express  the  result  in  percentage  of  SO3  simply  divide  the  value 
of  X,  as  just  determined,  by  the  quantity  of  urine  used. 

2.  Inorganic  Sulphates.- — Folhi's  Method. — Place  25  c.c.  of 
urine  and  100  c.c.  of  water  in  a  200-250  c.c.  Erlenmeyer  flask  and 
acidify  the  diluted  urine  with  10  c.c.  of  dilute  hydrochloric  acid  (i 
volume  of  concentrated  HCl  to  4  volumes  of  water).  In  case  the 
urine  is  dilute  50  c.c.  may  be  used  instead  of  25  c.c.  and  the  volume 
of  water  reduced  proportionately.  Add  10  c.c.  of  5  per  cent  barium 
chloride  slowly,  drop  by  drop,  to  the  cold  solution  and  from  this  point 
proceed  as  indicated  in  the  method  for  the  determination  of  Total 
Sulphates,  page  378. 

Calculate  the  quantity  of  inorganic  sulphates,  expressed  as  SO3,  in 
the  twenty-four-hour  urine  specimen. 

Calculation. — Calculate  according  to  the  directions  given  under 
Total  Sulphates,  above. 

3.  Ethereal  Sulphates. — Faun's  Method. — ^Place  125  c.c.  of 
urine  in  an  Erlenmeyer  flask  of  suitable  size,  dilute  it  with  75  c.c.  of 
water  and  acidify  the  mixture  with  30  c.c.  of  dilute  hydrochloric  acid 
(i  volume  of  concentrated  HCl  to  4  volumes  of  water).  To  the  cold 
solution  add  20  c.c.  of  a  5  per  cent  solution  of  barium  chloride,  drop 
by  drop.^     Allow  the  mixture  to  stand  about  one  hour,  then  filter 

since  such  manipulation  is  invariably  attended  by  mechanical  losses.  The  crucibles  should 
always  be  provided  with  lids  and  tight  bottoms  during  the  ignition.  In  case  porcelaia 
Gooch  crucibles,  whose  bottoms  are  not  pro\-ided  with  a  non-perforated  cap,  are  used, 
the  crucible  may  be  placed  upon  the  lid  of  an  ordinan,'  platinum  crucible  during  ignition. 
The  lid  should  be  supported  on  a  triangle,  the  crucible  placed  upon  the  lid  and  the  flame 
applied  to  the  improvised  bottom.  Ignition  should  be  complete  in  lo  minutes  if  no  organic 
matter  is  present. 

'  It  is  considered  preferable  by  many  investigators  to  express  all  sulphur  values  in. 
terms  of  S  rather  than  SO,. 

-  See  note  (2)  at  the  bottom  of  page  378. 


380  PHYSIOLOGICAL    CHEMISTRY. 

it  through  a  dry  fiUer  paper/  Collect  125  c.c.  of  the  tillrate  and  boil 
it  gently  for  at  least  one-half  hour.  Cool  the  solution,  filter  off  the 
precipitate  of  BaSO^.  wash,  dry  and  ignite  it  according  lo  the  direc- 
tions given  on  page  378. 

Calculation. — The  weight  of  the  BaSO^  precipitate  should  be 
multiplied  by  2  since  only  one-half  (125  c.c.)  of  the  total  volume  (250 
c.c.)  of  fluid  was  precipitated  by  the  barium  chloride.  The  remaining 
calculation  should  be  made  according  to  directions  gi\en  under  Total 
Sulphates,  page  378. 

Calculate  the  ([uantity  of  ethereal  sulphates,  expressed  as  SO3,  in 
the  twenty-four-hour  urine  specimen. 

4.  Total  Sulphur. — Benedicts  Method.'- — Ten  cubic  centimeters 
of  urine  are  measured  into  a  small  (7-8  c.c.)  porcelain  evaporating  dish 
and  5  c.c.^  of  Benedict's  sulphur  reagenf*  added.  The  contents  of  the 
dish  are  evaporated  over  a  free  flame  which  is  regulated  to  keep  the 
solution  just  below  the  boiling-point,  so  that  there  can  be  no  loss  through 
spattering.  When  dryness  is  reached  the  flame  is  raised  slightly  until 
the  entire  residue  has  blackened.  The  flame  is  then  turned  up  in  two 
stages  to  the  full  heat  of  the  bunsen  burner  and  the  contents  of  the  dish 
thus  heated  to  redness  for  ten  minutes  after  the  black  residue  {which  first 
fuses)  has  become  dry.  This  heating  is  to  decompose  the  last  traces  of 
nitrate  (and  chlorate).  The  flame  is  then  removed  and  the  dish  allowed 
to  cool  more  or  less  completely.  Ten  to  twenty  cubic  centimeters 
of  dilute  (1:4)  hydrochloric  acid  is  then  added  to  the  residue  in  the 
dish,  which  is  then  warmed  gently  until  the  contents  have  completely 
dissolved  and  a  perfectly  clear,  sparkling  solution  is  obtained.  This 
dissolving  of  the  residue  requires  scarcely  two  minutes.  With  the  aid 
of  a  stirring  rod  the  solution  is  washed  into"  a  small  I'^rU-nmeyer  flask, 
diluted  with  cold,  distilled  water  to  100  150  c.c,  10  c.c.  of  to  per 
cent  barium  chloride  solution  added  drop  ])y  droj),  and  thi-  solution 
allowed  to  stand  for  al)ou!  an  liour.  ll  is  then  shaken  uj)  and 
filtered  as  usual  through  a  weighed  (jooch  crucible. 

Calculation.      Make  the  calculation  according  lo  directions  given 

'This  [jrL-<i|>il:ilc  ((insists  of  ilic  inorj^iuiii  sul|)li;iU-s.  If  it  is  dcsiri'd,  this  HaSO,, 
|irf(i|)it;ilf  may  lie  (ollected  in  a  (looch  (  rucible  or  on  an  ordinary  (|uantilativt-  (ilter  paper 
anri  a  delcrniination  of  inorganic  sulijliates  made,  using  llic  same  ti'i  lini(|iic  as  that  siig- 
g(*slf(|  on  \>.  3,8.  In  lliis  way  we  are  enabled  lo  dclcniiinc  the  inorganii  ;iiid  (■llu-rea! 
sulphates  in  the  same  sample  of  urine. 

-  Benedict:  .lournal  of  liiutoj^ical  Chemistry,  V'J,  p.  ,i,(),\,  H)Oi). 

'  If  the  urine  is  < oik  entrated  the  (juantity  should  he  slightly  increased. 

'  ("rystalli/.ed  ( opper  nitrate,  sul|)hur-frcc  or  of   known  suljihur  ( ontcnt        200  grams 

Sodium  or  potassium  chlorate ^o  grams 

Distilhrd  water  to 1000  (.(  . 

''  Sometimes  the  po-celain  glaze  cracks  during  heating,  in  which  case  the  solnlioii 
should  be  filtered  into  the  (lask. 


urine:  quantitative  analysis.  381 

under  Total  Sulphates,  p.  378.      Calculate   the   quantity  of  sulphur. 
expressed  as  SO3  or  S,  present  in  the  twenty-four-hour  urine  specimen. 

5.  Total  Sulphur. — Osborne-Folin  Method. — Place  25  c.c.  of 
urine^  in  a  200-250  c.c.  nickel  crucible  and  add  about  3  grams  of 
sodium  peroxide.  Evaporate  the  mixture  to  a  syrup  upon  a  steam 
water-bath  and  heat  it  carefully  over  an  alcohol  flame  until  it  solidi- 
fies (15  minutes).  Now  remove  the  crucible  from  the  flame  and 
allow  it  to  cool.  Moisten  the  residue  with  1—2  c.c.  of  water/  sprinkle 
about  7-8  grams  of  sodium  peroxide  over  the  contents  of  the  crucible 
and  fuse  the  mass  over  an  alcohol  flame  for  about  10  minutes.  Allow 
the  crucible  to  cool  for  a  few  minutes,  add  about  100  c.c.  of  water  to 
the  contents  and  heat  at  least  one-half  hour  over  an  alcohol  flame 
to  dissolve  the  alkali  and  decompose  the  sodium  peroxide.  Next 
rinse  the  mixture  into  a  400-450  c.c.  Erlenmeyer  flask,  by  means  of  hot 
water,  and  dilute  it  to  about  250  c.c.  Heat  the  solution  nearly  to  the 
boiling-point  and  add  concentrated  hydrochloric  acid  slowly  until  the 
nickelic  oxide,  derived  from  the  crucible,  is  just  brought  into  solution.^ 
A  few  minutes  boiling  should  now  yield  a  clear  solution.  In  case  too 
little  peroxide  or  too  much  water  was  added  for  the  final  fusion  a  clear 
solution  will  not  be  obtained.  In  this  event  cool  the  solution  and 
remove  the  insoluble  matter  by  filtration. 

To  the  clear  solution  add  5  c.c.  of  very  dilute  alcohol  (about  18-20 
per  cent)  and  continue  the  boiling  for  a  few  minutes.  The  alcohol  is 
added  to  remove  the  chlorine  which  was  formed  when  the  solution 
was  acidified.  Add  10  c.c.  of  a  10  per  cent  solution  of  barium  chloride, 
slowly,  drop  by  drop,*  to  the  liquid.  Allow  the  precipitated  solution 
to  stand  in  the  cold  two  days  and  then  filter  and  continue  the  manipu- 
lation according  to  the  directions  given  under  Total  Sulphates, 
page  378. 

Calculation. — Make  the  calculation  according  to  directions  given 
under  Total  Sulphates,  p.  378.  Calculate  the  quantity  of  sulphur, 
expressed  as  SO3  or  S,  present  in  the  twenty-four-hour  urine  specimen. 

6.  Total  Sulphur. — Sodium  Hydroxide  and  Potassium  Nitrate 
Fusion  Method. — Place  25  c.c.  of  urine  in  a  silver  crucible  and  evap- 
orate to  a  thick  syrup  on  a  water-bath.  Add  10  grams  of  sodium 
hydroxide  and  2  grams  of  potassium  nitrate  to  the  residue  and  fuse 
the  mass,  over  an  alcohol  flame,  until  all  organic  matter  has  disap- 

'  If  the  urine  is  very  dilute  50  c.c.  may  be  used. 

-  This  moistening  of  the  residue  with  a  small  amount  of  water  is  very  essential  and 
should  not  be  neglected. 

^  About  18  c.c.  of  acid  is  required  for  8  grams  of  sodium  peroxide. 
*  See  note  (2)  at  the  bottom  of  page  378. 


382 


PHYSIOLOGICAL   CHEMISTRY. 


peared  and  the  fused  mixture  is  clear.  Cool  the  mixture,  transfer  it 
to  a  casserole  by  means  of  hot  water,  acidify  slightly  with  hydro- 
chloric acid  and  evaporate  it  to  dryness  on  a  water-bath.    Moisten  the 


Fig.  124.— Bkk  I II  1.1. 


(Ckoss-skction  of  Apparatus 


INSAIKK     JJO.VIH     (    AI.OKIMKTl'.K. 

AS  Rkady  for  Usk.) 

A.  Steel  f.uf)  or  Ijomh  i^rofjcr;  C,  collar  of  steel;  G,  oijening  llirough  which  oxygen  is 
forced  into  the  homh;  If  and  I',  insulated  wires  which  serve  to  conduct  an  electric  current 
for  igniting  the  substance  which  is  hekl  in  the  small  cap.sule;  Jj,  a  stirrer  which  serves  to 
keep  the  water  surrounrling  the  hf)mlj  in  motion  anrl  insures  the  ef|ualization  of  temper- 
ature; P,  a  delicate  thermometer  which  shows  the  rise  in  temperature  of  the  water  surround- 
ing the  bomb. 


residue  with  a  few  drops  (jf  dilute  hydnjchioric  acid  and  bring  it  into 
solution  with  hot  water.  Filter,  heat  the  fillralc  to  boiling,  and  imme- 
diately prcri[)itatc  it  by  the  addition  of  10  c.c.  of  a  10  per  cent  solution 


urine:  quantitative  analysis.  383 

of  barium  chloride,  adding  the  solution  slowly,  drop  by  drop.  Allow 
the  precipitated  solution  to  stand  2  hours  and  filter  while  cold.  Ignite, 
weigh,  and  calculate  according  to  directions  given  under  Total  Sul- 
phates, p.  378. 

Compute  the  quantity  of  sulphur,  expressed  as  SO3  or  S,  present 
in  the  twenty-four-hour  urine  specimen. 

7.  Total  Sulphur. — Sherman^ s  Compressed  Oxygen  Method} — 
Evaporate  as  much  urine  on  an  absorbent  filter  block^  at  55°  C.  as 
the  block  will  conveniently  absorb  and  burn  the  block  so  prepared 
in  a  bomb-calorimeter'  using  25-30  atmospheres  of  oxygen.  Connect 
the  bomb  with  a  wash-bottle  containing  water,  and  allow  the  gas  to 
bubble  through  the  liquid  until  the  high  pressure  within  the  appa- 
ratus has  been  reduced  to  atmospheric  pressure.  Nov»r  open  the  bomb 
and  thoroughly  rinse  the  interior,  using  water  from  the  wash-bottle 
for  the  first  rinsing.  Dissolve  any  ash  found  in  the  combustion  cap- 
sule in  hydrochloric  acid  and  add  this  solution  to  the  main  solution. 
Evaporate  to  150  c.c,  filter,  and  cool  the  filtrate.  Add  10  c.c.  of  a  5 
per  cent  solution  of  barium  chloride  to  the  cold  filtrate,  slowly,  drop 
by  drop.*  The  contents  of  the  flask  should  not  be  stirred  or  shaken 
during  the  addition  of  the  barium  chloride.  Allow  the  mixture  to 
stand  at  least  one  hour,  then  shake  up  the  solution  and  filter  it  through 
a  weighed  Gooch  crucible.  Manipulate  the  precipitate  of  BaSO^ 
according  to  directions  given  under  Total  Sulphates,  page  378. 

Calculate  the  quantity  of  sulphur,  expressed  as  SO3  or  S,  present 
in  the  twenty-four-hour  urine  specimen. 

IX.  Phosphorus. 

I.  Total  Phosphates. — Uranium  Acetate  Method. — To  50  c.c. 
of  urine  in  a  small  beaker  or  Erlenmeyer  flask  add  5  c.c.  of  a  special 
sodium  acetate  solution^  and  heat  the  mixture  to  the  boiling-point. 
From  a  burette,  run  into  the  hot  mixture,  drop  by  drop,  a  standard 
solution  of  uranium  acetate"  until  a  precipitate  ceases  to  form  and  a 

'  See  Sherman's  Organic  Analysis,  p.  19. 

^  Only  a  small  amount  of  urine  should  be  added  at  one  time,  it  being  necessary  to  make 
several  evaporations  before  the  block  contains  sufficient  urinary  residue  to  proceed  vt'ith 
the  combustion. 

^  The  Berthelot-Atwater  apparatus  (Fig.  124,  page  382)  is  well  adapted  to  this  purpose. 

^  See  note  (2)  at  the  bottom  of  page  378. 

^  The  sodium  acetate  solution  is  prepared  by  dissolving  100  grams  of  sodium  acetate 
in  800  c.c.  of  distilled  water,  adding  100  c.c.  of  30  per  cent  acetic  acid  to  the  solution,  and 
making  the  volume  of  the  mixture  up  to  i  liter  with  water. 

^This  uranium  acetate  solution  may  be  prepared  by  dissolving  35.461  grams  of 
uranium  acetate  in  one  liter  of  water.  One  c.c.  of  such  a  solution  should  be  equivalent  to 
0.005   gram  of  P2O5,   phosphoric  anhydride.     This  solution  may  be  standardized  as 


384  PHYSIOLOGICAL    CHEMISTRY. 

drop  of  the  mixture  when  removed  by  means  of  a  glass  rod  and  brought 
in  contact  with  a  drop  of  a  sokition  of  potassium  ferrocyanide  on  a 
porcelain  test-tablet  produces  instantaneously  a  brownish-red  colora- 
tion/ Take  the  burette  reading  and  calculate  the  P.,0-  content  of 
the  urine  under  examination. 

Calculation. — Multiply  the  number  of  cubic  centimeters  of  uranium 
acetate  solution  used  by  0.005  to  determine  the  number  of  grams  of 
PjOj  in  the  50  c.c.  of  urine  used.  To  express  the  result  in  percentage 
of  P,0-  multiply  the  value  just  obtained  by  2,  e.  g.,  if  50  c.c.  of 
urine  contained  0.074  gram  of  PjO^  it  would  be  equivalent  to  0.148 
per   cent. 

Calculate,  in  terms  of  PoO.,  the  total  phosphate  content  of  the 
twenty-four-hour   urine   specimen. 

2.  Earthy  Phosphates. — To  100  c.c.  of  urine  in  a  beaker  add  an 
excess  of  ammonium  hydroxide  and  allow  the  mixture  to  stand  12-24 
hours.  Under  these  conditions  the  phosphoric  acid  in  combination 
with  the  alkaline  earths,  calcium  and  magnesium,  is  precipitated  as 
phosphates  of  these  metals.  Collect  the  precipitate  on  a  filter  paper 
and  wash  it  with  very  dilute  ammonium  hydroxide.  Pierce  the  paper, 
and  remove  the  precipitate  by  means  of  hot  water.  Bring  the  phos- 
phates into  solution  by  adding  a  small  amount  of  dilute  acetic  acid  to 
the  warm  solution.  Make  the  volume  up  to  50  c.c.  with  water,  add  5 
c.c.  of  sodium  acetate  solution,  and  determine  the  P^O.  content  of  the 
mixture  according  to  the  directions  given  under  the  previous  method. 

Calculation. — Multiply  the  number  of  cubic  centimeters  of  uranium 
acetate  solution  used  by  0.005  ^^  determine  the  number  of  grams  of 
P^O.,  in  the  100  c.c.  of  urine  used.  Since  100  c.c.  of  urine  was  taken 
this  value  also  expresses  the  percentage  of  PjO.,  present. 

Calculate  the  ([uantity  oi  earthy  jjhosphates,  in  terms  of  P-.O^, 
present  in  the  twenty-four-hour  urine  sj)ecimen. 

The  quantity  of  phosphoric  acid  present  in  conil^ination  with  the 
alkali  metals  may  be  determined  by  subtracting  the  content  of  earthy 
phosphates  from   the  total   phosphates. 

Total  Phosphorus. — Sodium  Hydroxide  and  Potassium  Nitrate 
Fusion  Method.     Place  25  c.c.  of  urine  in  a  large  silver  crucible  and 

ffJIows:  IVj  50  c.c.  of  a  standard  sdIuiIoii  of  disodiuni  li\  (Iro^cii  |)li()S|)lialc,  ot  siK  li  a 
strength  that  the  .SOc.<:.  contains  o.  i  f^rairi  of  I'.,0.,,  add  5  < .(  .  of  the  sodium  a( ctatc  solution 
mc-ntionc-d  atjovc,  and  titrate  witli  the  uranium  solution  to  the  (orrect  end-reaction  as 
indi<ated  in  the  method  proper.  Inasmuch  as  i  c.c.  of  tlie  uranium  solution  should 
precipitate  0.005  K'"'*-'^  "^  ^' J h.<  exa(  tly  20  (  .( .  of  the  uranium  solution  should  he  re(|uircd 
to  |)re(  ipitate  50  c.c.  of  the  standarrl  jihosphale  solution.  If  the  two  solutions  do  not  bear 
this  relation  Ir)  each  father  ihey  may  he  brought  into  pro|)ri  relation  by  dilutin).^  the  uranium 
solution  with  distilled  water  or  hy  i.i(  reasing  its  streiif^lli. 

'  A  10  j>er  ( ent  solution  of  potassium  ferrocyanifle  is  satisfactory. 


urine:  quantitative  analysis.  385 

evaporate  to  a  syrup  on  a  water-bath.  Add  10  grams  of  NaOH  and 
2  grams  of  KNO3  to  the  residue  and  fuse  the  mass  until  all  organic 
matter  has  disappeared  and  the  fused  mixture  is  clear.  Cool  the  mix- 
ture, transfer  it  to  a  casserole  by  means  of  hot  water,  acidify  the  solu- 
tion slightly  with  pure  nitric  acid,  and  evaporate  to  dryness  on  a  water- 
bath.  Moisten  the  residue  with  a  few  drops  of  dilute  nitric  acid,  dis- 
solve it  in  hot  water,  and  transfer  to  a  beaker.  Now  add  an  equal 
volume  of  molybdic  solution^  and  keep  the  mixture  at  40°  C.  for 
twenty-four  hours.  Filter  off  the  precipitate,  wash  it  with  dilute 
molybdic  solution,  and  dissolve  it  in  dilute  ammonia.  Add  dilute 
hydrochloric  acid  to  the  solution,  being  careful  to  leave  the  solution 
distinctly  ammoniacal.  Magnesia  mixture^  (10-15  c.c.)  should  now 
be  added  and  after  stirring  thoroughly  and  making  strongly  ammoni- 
acal with  concentrated  ammonia  the  solution  should  be  allowed  to 
stand  in  a  cool  place  for  twenty-four  hours.  Filter  off  the  precipitate, 
wash  it  free  from  chlorine  by  means  of  dilute  ammonia  (1:5),  dry, 
incinerate,  and  weigh,  as  magnesium  pyrophosphate,  Mg^V^O^.  in  the 
usual  manner. 

In  this  method  the  phosphoric  acid  of  the  urine  is  precipitated  as 
ammonium  magnesium  phosphate  and  in  the  process  of  incineration 
this  body  is  transformed  into  magnesium  pyrophosphate. 

Calculation. — The  quantity  of  phosphorus,  expressed  in  terms  of 
P2O5,  in  the  volume  of  urine  taken  may  be  determined  by  means  of 
the   following  proportion: 

Mol.  wt.  Wt.  of  Mol.  wt. 

Mg,P,0,:Mg3P30,:  :P30,:^  (wt.  of  P,0,  in  grams), 
ppt. 

If  y  represents  the  weight  of  the  MgjPsO^  precipitate  and  we 
make  the  proper  substitution  we  have  the  following  proportion: 

221 .1 :}»:  :i4o.9:a'    (wt.    of    P2O5,    in    grams,    in    the    quantity    of 

urine  used.) 

To  express  the  result  in  percentage  of  P^O^  simply  divide  the  value 
of  X,  as  just  determined,  by  the  quantity  of  urine  used. 

X.  Creatinine. 

Folin's  Colorimetric  Method. — This  method  is  based  upon  the 
characteristic  property  possessed  alone  by  creatinine,  of  yielding  a 
certain  definite  color-reaction  in  the  presence  of  picric  acid  in  alkaline 

'  Directions  for  the  preparation  of  the  solution  are  given  on  p.  56. 

-  Directions  for  the  preparation  of  magnesia  mixture  may  be  found  on  p.  28c). 

25 


386  PHYSIOLOGICAL    CHEMISTRY. 

solution.  The  procedure  is  as  follows:  Place  10  c.c.  of  urine  in  a  500 
c.c.  volumetric  tiask.  add  15  c.c.  of  a  saturated  solution  of  picric  acid 
and  5  c.c.  of  a  10  per  cent  solution  of  sodium  hydroxide,  shake  thoroughly 
and  allow  the  mixture  to  stand  for  5  minutes.  During  this  interval 
pour  a  little  N  2  potassium  bichromate  solution^  into  each  of  the 
two  cylinders  of  the  colorimeter  (Duboscq's)  and  carefully  adjust  the 
depth  of  the  solution  in  one  of  the  cylinders  to  the  8  mm.  mark.  A 
few  preliminary  colorimetric  readings  may  now  be  made  with  the 
solution  in  the  other  cylinder,  in  order  to  insure  greater  accuracy  in 
the  subsequent  examination  of  the  solution  of  unknown  strength. 
Obviously  the  two  solutions  of  potassium  bichromate  are  identical  in 
color  and  in  their  examination  no  two  readings  should  differ  more 
than  0.1-0.2  mm.  from  the  true  value  (8  mm.).  Four  or  more  readings 
should  be  made  in  each  case  and  an  average  taken  of  all  of  them 
exclusive  of  the  iirst  reading,  which  is  apt  to  be  less  accurate  than  the 
succeeding  readings.  In  time  as  one  becomes  proficient  in  the 
technique  it  is  perfectly  safe  to  take  the  average  of  the  first  two 
readings. 

At  the  end  of  the  5-minute  interval  already  mentioned,  the  con- 
tents of  the  500  c.c.  flask  are  diluted  to  the  500  c.c.  mark,  the  bichro- 
mate solution  is  thoroughly  rinsed  out  of  one  of  the  cylinders,  and 
replaced  with  the  solution  thus  prepared  and  a  number  of  colorimetric 
readings  are  immediately  made. 

Ordinarily  10  c.c.  of  urine  is  used  in  the  determination  by  this 
method,  but  if  the  content  of  creatinine  is  above  15  mg.  or  below  5  mg. 
the  determination  should  be  repeated  with  a  volume  of  urine  selected 
according  to  the  content  or  creatinine.  This  variation  in  the  volume 
of  urine  according  to  the  content  of  creatinine  is  quite  essential,  since 
the  method  loses  in  accuracy  when  more  than  15  mg.  or  less  than  5  mg. 
of  creatinine  is  present  in  the  solution  of  unknown  strength. 

Calculation. — By  experiment  it  has  been  determined  that  10  mg. 
of  pure  creatinine,  when  brought  into  solution  and  diluted  to  500  c.c. 
as  explained  in  the  above  method,  yields  a  mixture  8.1  mm.  of  which 
possesses  the  same  colorimetric  value  as  8  mm.  of  a  N/2  solution  of  po- 
tassium bichromate.  Hearing  this  in  mind  ihe  computation  is  readily 
made  by  means  of  the  following  proportion  in  which  y  represents  the 
number  of  mm.  of  the  solution  of  unknown  strength  equivalent  lo  the 
8  mm.  of  the  potassium  bichromate  solution: 

y  :  8.  [   :  :  ID  :  .r  (mgs.  of  creatinine  in  Ihe  (|uanlily  of  urine  usedj. 

'  This  solution  lonta'ms  24.55  grams  of  |)otassiuiii  l)i(  liioniaU-  to  llic  lilcr. 


urine:  quantitative  analysis.  387 

This  proportion  may  be  used  for  the  calculation  no  matter  what 
volume  of  urine  (5,  10,  or  15  c.c.)  is  used  in  the  determination.  The 
10  represents  10  mg.  of  creatinine  which  gives  a  color  equal  to  8.1  mm., 
whether  dissolved  in  5,  10,  or  15  c.c.  of  fluid. 

Calculate  the  quantity  of  creatinine  in  the  twenty-four-hour  urine 
specimen. 

XI.  Creatine. 

Folin-Benedict  and  Myers  Method.^ — To  20  c.c.  of  urine  in  a 
50  c.c.  volumetric  flask,  add  20  c.c.  of  normal  hydrochloric  acid  and 
place  the  flask  in  an  autoclave  at  a  temperature  of  117—120°  C.  for 
one-half  hour.  Add  distilled  w^ater  until  the  volume  of  the  acid-urine 
mixture  is  exactly  50  c.c,  close  the  flask' by  means  of  a  stopper,  and 
shake  it  thoroughly.  Approximately  neutralize  25  c.c.  of  this  mixture, 
introduce  it  into  a  500  c.c.  volumetric  flask  and  determine  its  creatinine 
content  according  to  Folin's  Method  (see  p.  385). 

Calculation. — Calculate  as  explained  on  p.  386,  and  from  this 
\'alue  subtract  the  value  for  the  original  content  of  creatinine  before 
hydrolysis.  The  difference  between  these  two  values  will  be  the 
creatine  content  of  the  original  urine  in  terms  of  creatinine. 

XII.  Indican. 

EUinger's  Method. — This  method  for  the  quantitative  determin- 
ation of  indican  is  based  upon  the  principle  underlying  Jaffe  's  test  for 
the  detection  of  indican  (see  p.  275).  The  method  is  as  follows:  To 
50  c.c.  of  urine^  in  a  small  beaker  of  casserole  add  5  c.c.  of  basic  lead 
acetate  solution,  mix  thoroughly,  and  filter.  Transfer  40  c.c.  of  the 
filtrate  to  a  separatory  funnel,  add  an  equal  volume  of  Obermayer's 
reagent  (see  p.  275)  and  20  c.c.  of  chloroform,  and  extract  in  the  usual 
manner.  This  extraction  with  chloroform  should  be  repeated  until 
the  chloroform  solution  remains  colorless.  Now  filter  the  combined 
chloroform  extracts  through  a  dry  filter  paper  into  a  dry  Erlenmeyer 
flask.  Distil  off  the  chloroform,  heat  the  residue  on  a  boiling  water- 
bath  for  5  minutes  in  the  open  flask,  and  wash  the  dried  residue  with 
hot  water.^    Add  10  c.c.  of  concentrated  sulphuric  acid  to  the  washed 

^  Benedict  and  Myers:     Am.  J.  Phys.,  XVIII,  p.  397,  1907. 

■  If  the  urine  under  examination  is  neutral  or  alkaline  in  reaction  it  should  be  made 
faintly  acid  with  acetic  acid  before  adding  the  basic  lead  acetate. 

'  The  washing  should  be  continued  until  the  wash  water  is  no  longer  colored.  Ordi- 
narily two  or  three  washings  are  sufScient.  It  a  separation  of  indigo  particles  takes  place 
during  the  washing  process,  the  wash  water  should  be  filtered,  the  indigo  extracted  with 
chloroform,  and  the  usual  method  applied  from  this  point. 


388  PHYSIOLOGICAL    CHEMISTRY. 

residue,  heat  on  the  water-bath  for  5-10  minutes,  dilute  with  100  c.c. 
of  water,  and  titrate  the  bkie  solution  with  a  very  dilute  solution  of  po- 
tassium permanganate/  The  end-point  is  indicated  by  the  dissipation 
of  all  the  blue  color  from  the  solution  and  the  formation  of  a  pale 
yellow   color. 

Beautiful  plates  of  indigo  blue  sometimes  appear  in  the  chloroform 
extract  of  urines  containing  abundant  indican.  In  urines  preserved 
by  thymol  the  determination  of  indican  is  interfered  with.  Care 
should  be  taken,  therefore,  to  make  the  indican  determination  upon 
fresh  urine,  before  the  addition  of  the  preservative. 

Calculation. — EUinger  claims  that  one-sixth  of  the  amount  deter- 
mined must  be  added  to  the  value  obtained  by  titration  in  order  to 
secure  accurate  data.    This  correction  should  always  be  made. 

XIII.  Chlorides. 

T.  Clark's  Modification  of  Dehn's  Method.' — In  this  method 
the  organic  compounds,  that  hold  the  chlorine  too  firmly  for  its  quan- 
titative precipitation  with  argentic  nitrate,  are  destroyed  by  oxidation 
with  sodium  peroxide.  Sodium  peroxide  in  the  presence  of  water 
gives  off  nascent  oxygen  according  to  the  following  cc|uation: 

Na202  +  H20  =  2NaOH  +  0. 

The  oxygen  then  attacks  the  organic  matter  and  the  chlorine  is 
left  as  sodium  chloride.  The  procedure  is  as  follows:  To  10  c.c.  of 
urine  in  a  75-100  c.c.  casserole,  add  i. 0-1.2  gram  of  sodium  peroxide 
and  evaporate  the  mixture  to  dryness  on  a  Ijoiling  water-bath.  In 
case  the  residue  is  not  pure  white,  thus  indicating  that  insufficient 
sodium  peroxide  has  been  added,  the  residue  should  be  moistened 
with  distilled  water,  additional  sodium  peroxide  added,  and  the  mix- 
ture again  evaporated  to  dryness.  When  the  oxidation  is  complete, 
treat  the  mass  with  10-20  c.c.  of  distilled  water  and  slir  until  it  has 
practically  all  been  brought  into  solution.  Then  introduce  a  bit  of  litmus 
pa[jer  and  add  dilute  nitric  acid  (i  :i)  until  the  litmus  ])aper  turns  red 
and  (ill  effervescence  ceases.  Now  jjJace  the  casserole  on  a  hot  plate 
or  on  a  gauze  and  heal  the  conlcnls  almost  to  the  boiling  point. ^     To 

'  A  "slock  solution"  of  potassium  jjcrmanj^anale  containing  .^  grams  per  liter 
should  be  preparefl,  and  when  nccfled  for  titration  [)urjjoses  a  suitaljle  volume  of  this 
solution  should  be  flilulcrl  with  40  volumes  of  water.  The  [Milassium  permanganate 
s'>lution  should  be  standardized  with  pure  indigo. 

^Private  communication  to  the  author  from  Mr.  S.  C.  Clark. 

'  ff  there  is  a  slight  precipitate,  flue  to  silicic  acid  from  the  casserole,  this  is  liilered 
of!  and  the  fdtrate  collected  in  a  200  c.c.  I)eaker. 


urine:  quantitative  analysis.  389 

the  hot  solution  add  a  standard  solution  of  argentic  nitrate  (see  page 
390)  in  slight  excess.^  Filter  off  the  silver  chloride  while  the  solution 
is  still  hot  and  wash  the  precipitate  thoroughly  with  distilled  water. 
To  the  filtrate,  add  i  c.c.  of  a  saturated  solution  of  ferric  ammonium 
sulphate  and  then  titrate  with  a  standard  solution  of  ammonium 
thiocyanate  (see  page  390)  until  the  clear,  slightly  yellow  fluid  (or  the 
opalescent,  milky  fluid,  in  case  there  is  much  excess  of  argentic  nitrate) 
changes  to  a  slight  reddish-brown  color.  The  color  of  the  end-point 
varies  with  the  indi\idual.  The  exact  end-point  reached  is  not  so 
important  as  is  the  securing  of  the  same  end-point  in  a  series  of  deter- 
minations as  that  obtained  in  the  standardization  of  the  standard 
solutions  used. 

Calculation. — The  standard  solution  of  argentic  nitrate  should  be 
made  up  so  that  i  c.c.  equals  o.oio  gram  of  sodium  chloride  and  i 
c.c.  of  the  ammonium  thiocyanate  should  be  equivalent  to  i  c.c.  of 
the  argentic  nitric  solution  (see  p.  390).  Then,  if  the  number 
of  cubic  centimeters  of  ammonium  thiocyanate  used  be  subtracted 
from  the  number  of  cubic  centimeters  of  argentic  nitrate,  the  dif- 
ference is  the  number  of  cubic  centimeters  of  argentic  nitrate  actually 
used  in  the  precipitation  of  chlorine  as  silver  chloride.  This  num- 
ber, multiplied  by  o.oio,  gives  the  weight  in  grams  of  the  sodium 
chloride  in  the  10  c.c.  of  urine  used.  If  it  is  desired  to  express  the 
result  in  percentage  of  sodium  chloride,  move  the  decimal  point  one 
place  to  the  right. 

In  a  similar  manner  the  weight  or  percentage  of  chlorine  may  be 
computed,  using  the  factor  0.006  as  explained  in  Mohr's  method, 
below.  Calculate  the  quantity  of  sodium  chloride  and  of  chlorine 
in  the  twenty-four-hour  urine  specimen. 

2.  Mohr's  Method. — To  10  c.c.  of  urine  in  a  small  platinum  or 
porcelain  crucible  or  dish  add  about  2  grams  of  chlorine-free 
potassium  nitrate  and  evaporate  to  dryness  at  100°  C.  (The  evapora- 
tion may  be  conducted  over  a  low  flame  provided  care  is  taken  to  pre- 
vent loss  by  spurting.)  By  means  of  crucible  tongs  hold  the  crucible  or 
dish  over  a  free  flame' until  all  carbonaceous  matter  has  disappeared 
and  the  fused  mass  is  slightly  yellow  in  color.  Cool  the  residue  some- 
what and  bring  it  into  solution  in  a  small  amount  (15-25  c.c.)  of  dis- 
tilled water  acidified  with  about  10  drops  of  nitric  acid.  Transfer 
the  solution  to  a  small  beaker,  being  sure  to  rinse  out  the  crucible  or 
dish  very  carefully.     Test  the  reaction  of  the  fluid,  and  if  not  already 

*  This  point  is  most  easily  recognized  by  keeping  the  solution  hot  and  in  constant 
agitation  while  adding  the  argentic  nitrate  so  that  the  silver  chloride  formed  coagulates  and 
sinks,  leaving  a  clear,  supernatant  fluid. 


390 


PHYSIOLOGICAL    CHEMISTRY. 


acid  in  reaction  to  litmus,  render  it  slightly  acid  with  nitric  acid.  Now 
neutralize  the  solution  by  the  addition  of  calcium  carbonate^  in  sub- 
stance, add  2-5  drops  of  neutral  potassium  chromate  solution  to  the 
mixture,  and  titrate  with  a  standard  argentic  nitrate  solution.- 

This  standard  solution  should  be  run  in  from  a  burette,  stirring 
the  liquid  in  the  beaker  after  each  addition.  The  end-reaction  is 
reached  when  the  yellow  color  of  the  solution  changes  to  a  slight  orange- 
red.  At  this  point  take  the  burette  reading  and  compute  the  per- 
centage of  chlorine  and  sodium  chloride  in  the  urine  examined. 

Calculation. — Since  i  c.c.  of  the  standard  argentic  nitrate  solu- 
tion is  equivalent  to  o.oio  gram  of  sodium  chloride,  to  obtain  the 
weight,  in  grams,  of  the  sodium  chloride  in  the  10  c.c.  of  urine  used 
multiply  the  number  of  cubic  centimeters  of  standard  solution  used 
by  O.OIO.  If  it  is  desired  to  express  the  result  in  percentage  of  sodium 
chloride  move  the  decimal  point  one  place  to  the  right. 

To  obtain  the  weight,  in  grams,  of  the  chlorine  in  the  10  c.c.  of 
urine  used  multiply  the  number  of  cubic  centimeters  of  standard 
solution  used  by  0.006,  and  if  it  is  desired  to  express  the  result  in  per- 
centage of  chlorine  move  the  decimal  point  Ofie  place  to  the  right. 

Calculate  the  quantity  of  sodium  chloride  and  chlorine  in  the 
twenty-four-hour  urine  specimen. 

3.  Volhard-Arnold  Method. — Place  10  c.c.  of  urine  in  a  100 
c.c.  volumetric  flask,  add  20-30  drops  of  nitric  acid  (sp.  gr.  1.2)  and 
2  c.c.  of  a  cold  saturated  solution  of  ferric  alum.  If  necessary,  at  this 
point  a  few  drops  of  an  8  ])er  cent  solution  of  potassium  permanga- 
nate may  be  added  to  dissipate  the  red  color.  Now  slowly  run  in 
the  standard  argentic  nitrate^  solution  (20  c.c.  is  ordinarily  used) 
until  all  the  chlorine  has  iK-en  j)rcci])itated  and  an  excess  of  the  argentic 
nitrate  solution  is  jjresent,  continually  shaking  the  mixture  during  the 
addition  of  the  standard  solution.  Allow  the  llask  to  stand  lo  minutes, 
then  fill  it  to  the  100  c.c.  graduation  with  distilled  water  and  thoroughly 
mix  the  contents.  Now  filter  the  mixture  through  a  dry  filter  pa])er, 
collect  50  c.c.  of  the  filtrate  and  titrate  il  with  standardized  ammonium 
thiocyanate  solution. "''     The  first  permanent  tinge  of  brown  indicates 


'  The  cessation  of  effervescence  and  llie  presence  of  some  undeconiixjsecl  c;ilciuin 
carbonate  at  the  bottom  of  the  vessel  arc  the  indications  of  neutralization. 

^Standard  argentic  nitrate  solution  may  be  prepared  by  dissolving  29.060  grams  of 
argentic  nitrate  in  i  liter  of  distilled  water.  Each  cul)ic  centimeter  01  this  solution  is 
equivalent  to  o. 010 gram  of  smlium  chloride  or  to  0.006  gram  of  chlorine. 

'  This  solution  is  made  of  such  a  strength  that  1  <  .<:.  of  it  is  e(jual  to  r  c.c.  of  the  standard 
argentic  nitrate  solution  used.  Tf)  jjrejiare  the  solution  rlissolve  12.9  grams  of  ammonium 
thiocyanate,  .\U,SC.V,  in  a  little  less  than  a  liter  of  water.  In  a  small  llask  place  20  c.c. 
of  the  .standard  argentic  nitrate  sf>liilion,   i,  (  .c.  of  tin-  ferric  alum  solution  and  4  c.c.  of 


urine:  quantitative  analysis.  391. 

the  end-point.  Take  the  burette  reading  and  compute  the  weight 
of  sodium  chloride  in  the  10  c.c.  of  urine  used. 

Calculation. — The  number  of  cubic  centimeters  of  ammonium 
thiocyanate  solution  used  indicates  the  excess  of  standard  argentic 
nitrate  solution  in  the  50  c.c.  of  filtrate  titrated.  Multiply  this  reading 
by  2,  inasmuch  as  only  one-half  of  the  filtrate  was  employed,  and 
subtract  this  product  from  the  number  of  cubic  centimeters  of  argentic 
nitrate  (20  c.c.)  originally  used,  in  order  to  obtain  the  actual  number 
of  cubic  centimeters  of  argentic  nitrate  utilized  in  the  precipitation  of 
the  chlorides  in  the  10  c.c.  of  urine  employed. 

To  obtain  the  weight  in  grams  of  the  sodium  chloride  in  the 
10  c.c.  of  urine  used,  multiply  the  number  of  cubic  centimeters  of 
the  standard  argentic  nitrate  solution,  actually  utilized  in  the  pre- 
cipitation, by  o.oio.  If  it  is  desired  to  express  the  result  in  per- 
centage of  sodium  chloride  move  the  decimal  point  one  place  to  the 
right. 

In  a  similar  manner  the  weight,  or  percentage  of  chlorine  may 
be  computed  using  the  factor  0.006  as  explained  in  Mohr's  method, 
page  389. 

Calculate  the  quantity  of  sodium  chloride  and  chlorine  in  the 
twenty-four-hour  urine  specimen. 

XIV.  Acetone  and  Diacetic  Acid. 

I.  Folin-Hart  Method. — This  method  serves  the  same  purpose 
as  the  Messinger-Huppert  Method,  i.  e.,  the  determination  of  both 
acetone  and  diacetic  acid  in  terms  of  acetone.  It  is,  however,  much 
simpler  and  less  time-consuming.  The  method  includes  the  trans- 
formation of  the  diacetic  acid  into  acetone  and  carbon  dioxide  by 
means  of  heat  and  the  subsequent  removal  of  the  acetone  thus  formed, 
as  well  as  the  preformed  acetone,  by  means  of  an  air  current  as  first 
suggested  by  Fohn  (see  p.  393).  The  procedure  is  as  follows:  Intro- 
duce into  a  wide-mouthed  bottle  200  c.c.  of  w^ater,  an  accurately 
measured  excess  of  N/io  iodine  solution^  and  an  excess  of  40  per  cent 

nitric  acid  (sp.  gr.  i .  2),  add  water  to  make  the  total  volume  100  c.c.  and  thoroughly  mix 
the  contents  of  the  flask.  Now  run  in  the  ammonium  thiocyanate  solution  from  a  burette 
until  a  permanent  brown  tinge  is  produced.  This  is  the  end-reaction  and  indicates  that 
the  last  trace  of  argentic  nitrate  has  been  precipitated.  Take  the  burette  reading  and 
calculate  the  amount  of  water  necessary  to  use  in  diluting  the  ammonium  thiocyanate  in 
order  that  10  c.c.  of  this  solution  may  be  exactly  equal  to  10  c.c.  of  the  argentic  nitrate 
solution.  Make  this  dilution  and  titrate  again  to  be  certain  that  the  solution  is  of  the  proper 
strength. 

'  Proceed  as  follows  in  order  to  obtain  a  rough  idea  regarding  the  amount  of  X  10  iodine 
solution  to  be  used:  Introduce  into  a  test-tube  10  c.c.  of  the  urine  under  examination 
and  I  c.c.  of  a  solution  of  ferric  chloride  made  by  dissoh-ing  100  grams  of  ferric  chloride 


392  PHYSIOLOGICAL    CHEMISTRY. 

potassium  hydroxide.  Prepare  an  aerometer  cylinder  containing 
alkaline  hypoiodite  solution  to  absorb  any  acetone  which  may  be  pres- 
ent in  the  air  of  the  laboratory,  and  between  the  cylinder  and  bottle 
suspend  a  test-tube  about  two  inches  in  diameter.  This  large  test- 
tube  should  contain  20  c.c.  of  the  urine  under  examination,  10  drops 
of  a  10  per  cent  solution  of  phosphoric  acid,  10  grams  of  sodium 
chloride,  and  a  little  petroleum,  and  should  be  raised  sufficiently 
high  to  facilitate  the  easy  application  of  heat  to  its  bottom  portion. 
The  connections  on  the  side  of  the  tube  should  be  provided  with  bulb- 
tubes  containing  cotton.  When  the  apparatus  is  arranged  as  described, 
it  should  be  connected  with  a  Chapman  pump  and  an  air  current  passed 
through  for  twenty-five  minutes.  During  this  period  the  contents  of 
the  test-tube  are  heated  just  to  the  boiling-point  and  after  an  interval 
of  five  minutes  again  heated  in  the  same  manner.  By  this  means  the 
diacetic  acid  is  converted  into  acetone  and  at  the  end  of  the  twenty- 
fivc-minute  period  this  acetone,  as  well  as  the  preformed  acetone,  will 
have  been  removed  from  the  urine  to  the  absorption  bottle  and  there 
retained  as  iodoform. 

The  contents  of  the  absorption  bottle  should  now  be  acidified  with 
concentrated  hydrochloric  acid,^  and  titrated  with  N/io  sodium  thio 
sulphate  and  starch  as  in  the  Messinger-Huppert  method  (see  below). 

2.  Messinger-Huppert  Method.^ — Place  100  c.c.  of  urine  in  a 
distillation  flask  and  add  2  c.c.  of  50  per  cent  acetic  acid.  Connect 
the  flask  with  a  condenser,  properly  arrange  a  receiver,  attach  a 
terminal  series  of  bulbs  containing  water,  and  distil  over  about  nine- 
tenths  of  the  urine  mixture.  Remove  the  receiver,  attach  another, 
and  subject  the  residual  portion  of  the  mixture  to  a  second  distil- 
lation.    Test  this  fluid  for  acetone  and  if  the  presence  of  acetone 

in  100  c.c.  of  distilled  water.  After  permitting  tlie  mixture  to  stand  for  two  minutes, 
compare  the  cf)lor  with  that  of  an  equal  volume  of  the  ferric  chloride  .solution  in  a  test-tube 
of  similar  rliameter.  If  the  two  solutions  he  of  approximately  the  same  color  intensity, 
20  c.c.  of  the  urine  under  examination  will  3'ield  suHicient  acetone  to  require  nearly  lo  c.c. 
of  X/io  iodine  solution.  In  case  the  mixture  is  darker  in  color  than  is  the  ferric  chloride 
sf)lutif>n,  the  former  should  he  diluted  with  distilled  water  until  it  is  of  approximately  the 
.siimc  intensity  as  the  ferric  chloride  solution.  From  this  data  the  amount  of  N/io  iodine 
scjjution  require<l  may  he  roughly  estimated  by  means  of  the  following  table: 

unne  c.c  I'crrK  i  lilondc.  Water.  '  ' 


10 

1 

10 

ro 

1          1                 10 

20 

10 

1                 20 

35 

10 

I                 30 

5° 

'  An  excess  of  iodine  is  indicated  by  the  develojjment  of  a  brown  color. 

'''  This  methofi  'icrvf^  to  determine  bolh  acet<jne  and  diacetic  acid  in  terms  of  acetone. 


urine:  quantitative  analysis.  393 

is  indicated  add  about  loo  c.c.  of  water  to  the  residue  and  again 
distil.  Treat  the  united  acetone  distillates  with  i  c.c.  of  dilute  (12 
per  cent)  sulphuric  acid  and  redistil,  collecting  this  second  distillate 
in  a  glass-stoppered  flask.  During  distillation,  however,  the  glass 
stopper  is  replaced  by  a  cork  with  a  double  perforation,  the  glass 
tube  from  one  perforation  passing  to  the  condenser,  while  the  bulbs 
containing  water,  before  mentioned,  are  attached  by  means  of  the 
tube  in  the  other  perforation.  Allow  the  distillation  process  to  pro- 
ceed until  practically  all  of  the  fluid  has  passed  over,  then  remove 
the  receiving  flask  and  insert  the  glass  stopper.  Now  treat  the  dis- 
tillate carefully  wdth  10  c.c.  of  a  N/io  solution  of  iodine  and  add  sodium 
hydroxide  solution,  drop  by  drop,  until  the  blue  color  is  dissipated 
and  the  iodoform  precipitates.  Stopper  the  flask  and  shake  it  for 
about  one  minute,  acidify  the  solution  with  concentrated  hydrochloric 
acid,  and  note  the  production  of  a  brown  color  if  an  excess  of  iodine 
is  present.  In  case  there  is  no  such  excess,  the  solution  should  be 
treated  with  N/io  iodine  solution  until  an  excess  is  obtained.  Reti- 
trate  this  excess  of  iodine  with  N/io  sodium  thiosulphate  solution 
until  a  light  yellow  color  is  observed.  At  this  point  a  few  cubic  centime- 
ters of  starch  paste  should  be  added  and  the  mixture  again  titrated 
until  no  blue  color  is  visible.     This  is  the  end-reaction. 

Calculation. — Subtract  the  number  of  cubic  centimeters  of  N/io 
thiosulphate  solution  used  from  the  volume  of  N/io  iodine  solution 
employed.  Since  i  c.c.  of  the  iodine  solution  is  equivalent  to  0.967 
milligram  of  acetone,  and  since  i  c.c.  of  the  thiosulphate  solution  is 
equivalent  to  i  c.c.  of  the  iodine  solution,  if  we  multiply  the  remainder 
from  the  above  subtraction  by  0.967  we  will  obtain  the  number  of 
milligrams  of  acetone  in  the  100  c.c.  of  urine  examined. 

Calculate  the  quantity  of  acetone  in  the  twenty-four-hour  urine 
specimen. 

XV.  Acetone. 

I.  Folin's  Method. — The  same  type  of  apparatus  is  used  in  this 
method  as  that  described  in  Folin's  method  for  the  determination 
of  ammonia  (see  p.  374).  The  procedure  is  as  follows:  Introduce 
20-25  c.c.  of  the  urine  under  examination  into  the  aerometer  cylinder 
and  add  10  drops  of  10  per  cent  phosphoric  acid,^  8-10  grams  of 
sodium  chloride,^  and  a  little  petroleum.     Introduce  into  an  absorp- 

'  Oxalic  acid  (0.2-0.3  o'^^.m)  ma}'  be  substituted  if  desired. 
-  Acetone  is  insoluble  in  a  saturated  solution  of  sodium  chloride. 


394  PHYSIOLOGICAL    CHEMISTRY. 

tion  flask/  such  as  is  used  in  the  ammonia  determinalion  (^see  p. 
374),  150  c.c.  of  water,  10  c.c.  of  a  40  per  cent  sokition  of  potassium 
hydroxide,  and  an  excess  of  a  N/ 10  iodine  sohition.  Conned  the  flask 
with  the  aerometer  cyhnder.  attach  a  Chapman  pump,  and  permit  an 
air  current,  slightly  less  rapid  than  that  used  for  the  determination 
of  ammonia,  to  be  drawn  through  the  solution  for  20-25  minutes. 
All  of  the  acetone  will,  at  this  point,  have  been  converted  into  iodo- 
form in  the  absorption  flask.  Add  10  c.c.  of  concentrated  hydro- 
chloric acid  (a  volume  equivalent  to  that  of  the  strong  alkali  orig- 
inally added),  to  the  contents  of  the  latter  and  titrate  the  excess  of  iodine 
by  means  of  X/io  sodium  thiosulphate  solution  and  starch,  as  in  the 
Messinger-Huppert  method  (see  p.  392). 

Folin  has  further  made  suggestions  regarding  the  simultaneous 
determination  of  acetone  and  ammonia  by  the  use  of  the  same  air 
current.-  This  is  an  important  consideration  for  the  clinician  inas- 
much as  urines  which  contain  acetone  and  diacetic  acid  are  gener- 
ally those  from  which  the  ammonia  data  are  also  desired.  The  pro- 
cedure for  the  combination  method  is  as  follows:  Arrange  the 
ammonia  apparatus  as  usual  (sec  p.  374),  and  to  the  aerometer  of  the 
ammonia  apparatus  attach  the  acetone  apparatus  set  up  as  described 
above.  Regulate  the  air  current  with  special  reference  to  the  deter- 
minalion of  acetone  and  at  the  end  of  20-25  minutes  disconnect  the 
acetone  apparatus  and  complete  the  determination  of  the  acetone  as 
just  described.  The  air  current  is  not  interrupted,  and  after  ha\ing 
run  one  and  one-half  hours  the  ammonia  apparatus  is  detached  and 
the  ammonia  determination  completed  as  described  on  page  374. 

If  data  regarding  diacetic  acid  are  desired,  the  result  obtained  by 
Folin's  method  may  be  subtracted  from  the  result  obtained  by  the 
Messinger-Huppert  method  (see  p.  392),  inasmuch  as  the  latter 
method  determines  both  acetone  and  diacetic  acid.  Under  all  condi- 
tions the  determination  of  acetone  should  be  as  expeditious  as  possiI)k'. 
This  is  essential,  not  only  l)ecause  of  the  fact  thai  any  diacetic  acid 
present  in  the  urine  will  become  transformed  into  acetone,  but  also 
because  of  the  rapid  spontaneous  decomposition  of  the  alkaline  hy]K)- 
ioditc  soluti(;n  used  in  the-  fjetermination  of  the  acetone.  It  has  been 
claimed  that  alkaline  hyjx^iodite  solutions  are  alniosl  completely 
convcrtcfl   into  imlalr  solutions  in   one-half  hour.     l''olin   slates,   hovv- 

'  Folin's  improved  ;il)S()r|)iion  mix:  (sec  I-'ig.  12.^,  p.  375)  should  he  iisfd  in  this  con- 
neclion  inasmuch  as  the  original  ty|)e  cmhracin^  the  use  of  a  ruhiicr  stopper  is  unsatis- 
factory tjccause  of  the  solvent  action  of  alkaline  hypoiodilf  on  niiihcr. 

^  These  fjeterminalions  may  even  be  made  on  the  \iiiiir  \iiiii/'/r  ni  mine  if  die  sample; 
is  too  small  for  the  df>uljle  delerminalion. 


urine:  quantitative  analysis.  395 

ever,  that  the  transformation  is  not  so  rapid  as  this,  but  he  nevertheless 
emphasizes  the  necessity  of  rapidity  of  manipulation.  At  the  same 
time  it  should  be  remembered  that  the  air  current  must  not  be  as  rapid 
as  for  ammonia,  inasmuch  as  the  alkaline  hypoiodite  solution  will  not 
absorb  all  the  acetone  under  those  conditions. 


XVI.  Diacetic  Acid. 

1.  Folin-Hart  Method. — Arrange  the  apparatus  as  described 
under  the  Folin-Hart  method  for  the  determination  of  acetone  and 
diacetic  acid  (see  p.  391).  Start  the  air  current  in  the  usual  way  and 
permit  it  to  run  25  minutes  without  the  application  of  heat  to  the  urine 
under  examination.  Under  these  conditions  the  preformed  acetone 
present  in  the  solution  is  all  removed  (see  p.  393).  Immediately  attach 
a  freshly  prepared  absorption  bottle  or  introduce  fresh  alkaline  hypo- 
iodite solution  into  the  original  bottle.  Apply  heat  to  the  large  test-tube 
as  already  described  (see  p.  392),  in  order  to  convert  the  diacetic  acid 
into  acetone,  permit  the  air  current  to  continue  for  the  usual  25-minute 
period,  and  determine  the  diacetic  acid  value  in  terms  of  acetone  by 
the  usual  titration  procedure  (see  p.  394). 

2.  Folin-Messinger-Huppert  Method.^Determine  the  com- 
bined acetone  and  diacetic  acid,  in  terms  of  acetone,  by  the  Messinger- 
Huppert  method  (see  p.  392),  and  subsequently  determine  the  acetone 
by  Folin's  method  (see  p.  393).  Subtract  the  value  determined  by 
the  second  method  from  that  obtained  in  the  first  method  to  secure 
data  regarding  the  diacetic  acid  content  of  the  urine,  in  terms  of  acetone. 

XVII.  /?-Oxybutyric  Acid. 

I.  Shaffer's  Method. — Introduce  25-250  c.c.  of  urine^  into  a 
500  c.c.  volumetric  flask  and  add  an  excess  of  basic  lead  acetate  and 
10  c.c.  of  concentrated  ammonium  hydroxide.  Dilute  the  mixture 
to  the  500  c.c.  mark,  shake  the  flask  thoroughly  and  filter.  Transfer 
200  c.c.  of  the  filtrate  to  an  800  c.c.  Kjeldahl  distilling  flask,  add 
300-400  c.c.  of  water,  15  c.c.  of  concentrated  sulphuric  acid  and  a 
little  talcum  and  distil  the  mixture  until  200  to  250  c.c.  of  distillate 

^  The  amount  used  depends  upon  the  expected  yield  of  f3-oxybutyric  acid.  In  the 
case  of  urines  which  give  a  strong  ferric  chloride  reaction  for  diacetic  acid,  or  when  5-ic 
grams  or  more  of  /?-ox}-butyric  acid  is  expected,  it  is  unnecessary  to  use  more  than  25-50 
G.c.  of  urine.  However,  in  case  only  a  trace  of  ,3-ox}'butyric  acid  is  expected,  the  volume 
should  be  much  larger  as  indicated.  Under  all  conditions,  the  amount  specified  is  suflicient 
for  dupHcate  determinations.  It  is  desirable  to  use  such  a  volume  of  urine  as  contains 
the  proper  amount  of  f3-oxybutyric  acid  to  yield  25-50  milligrams  of  acetone. 


396  PHYSIOLOGICAL    CHEMISTRY. 

has  been  collected  (A)/  To  this  distillate  (A),  which  contains  acetone 
i^both  preformed  and  that  produced  from  diacetic  acid),  and  volatile 
fatty  acids  is  added  5  c.c.  of  10  per  cent  potassium  hydroxide  and  the 
distillate  redistilled  in  order  to  remove  the  volatile  fatty  acids. ^  This 
second  distillate  (A,)  is  then  titrated  with  standard  iodine  and  thio- 
sulphate  (see  p.  394).  The  urine-sulphuric  acid  residue  from  which 
distillate  A  was  obtained  is  again  distilled,  400-600  c.c.  of  a  o.  1-0.5 
per  cent  potassium  bichromate  solution  being  added,  by  means  of  the 
dropping  tube,  during  the  process  of  distillation.^  In  adding  the 
bichromate,  care  should  be  taken  not  to  add  it  faster  than  the  distillate 
collects  except  in  cases  where  the  boiling  fluid  assumes  a  pure  green 
color,  thus  indicating  that  the  bichromate  is  being  used  up  more 
rapidly.  After  about  500  c.c.  of  distillate  (B)  has  collected,  20  c.c.  of 
a  3  per  cent  solution  of  hydrogen  peroxide  and  a  few  cubic  centimeters 
of  potassium  hydroxide  solution  are  added  and  the  mixture  (B)  sub- 
jected to  redistillation.  Distil  off  about  300  c.c.  and  titrate  this  dis- 
tillate (B,)  as  usual  with  iodine  and  thiosulphate  (see  p.  394). 

Calculation. — ^The  author  advises  the  use  of  solutions  of  thiosul- 
phate and  iodine,  which  are  a  trifle  stronger  than  N/ 10;  i.  e.,  103 . 4  N/ 10. 
Each  cubic  centimeter  of  an  iodine  solution  of  this  strength  is  equiva- 
lent to  one  milligram  of  acetone  or  to  i .  794  milligrams  of  /3-oxybutyric 
acid.  The  thiosulphate  solution  is  accepted  as  the  standard  and 
should  be  restandardizcd,  from  time  to  time,  by  a  N/io  solution  of 
potassium  h)i-iodate. 

2.  Black's  Method. — Render  50  c.c.  of  the  urine  under  examina- 
tion, faintly  alkaline  with  sodium  carbonate  and  evaporate  to  one- 
third  the  original  volume.  Concentrate  to  about  10  c.c.  on  a  water- 
bath,  cool  the  residue,  acidify  it  with  a  few  drops  of  concentrated  hy- 
drochloric acid*  and  add  plaster  of  Paris  to  form  a  thick  paste.  Per- 
mit the  mixture  to  stand  until  it  begins  to  "set,"  then  break  it  up  with 
a  stout  glass  rod  having  a  blunt  end  and  reduce  the  material  to  the 
consistency  of  a  fairly  dry  coarse  meal.^  Transfer  the  meal  to  a 
Soxhlet  apparatus  and  extract  with  ether  for  two  hours.  At  the  end 
of  this  period  evaporate  the  ether-extract  either  spontaneously  or  in 

'  This  distilling  flask  should  be  provirk-r|  wilii  ,i  <li()|)|)iii)^  tube,  by  means  of  which 
water  may  be  introduced  in  order  to  prevent  the  contents  of  liie  flask  from  becoming  loss 
than  400  c.c.  in  volume.  Care  should  be  taken  to  use  a  good  condenser  in  the  (lislilLill'in, 
but  it  is  not  necessary  to  cool  the  flistillate  with  ice. 

^  Formic  a<  id  is  one  of  the  most  troublesome. 

•' f lenerally  the  addition  of  o.';  gram  of  potassium  bi(  hnjiiialc  is  snllKieDl.  In  case 
the  urine  contains  a  high  concentratif)n  of  sugar  or  when  a  large  vdiimic  of  urine  is  used, 
it  may  be  necessary  to  use  2-^  grams  of  the  birhromalcs 

*  The  residue  shoulfl  give  a  distinct  rcfl  cdU^r  with  litmus  paper. 

'Before  this  is  a<(om[ilishefl  it  may,  in  some  cases,  be  necessary  to  .I'lii  :i  litllc  more 
plaster  of  fari*;. 


urine:  quantitative  analysis.  397 

an  air  current.  Dissolve  the  residue  in  water,  add  a  little  bone-black, 
if  necessary,  filter  until  a  clear  solution  is  obtained  and  make  up  the 
filtrate  to  a  known  volume  (25  c.c.  or  less)  with  water.  The  ,5-oxy- 
butyric  acid  should  then  be  determined  by  means  of  the  polariscope. 

3.  Darmstadter's  Method. — This  method  is  based  on  the  fact 
that  crotonic  acid  is  formed  from  /?-oxybutyric  acid  under  the  influence 
of  concentrated  mineral  acids.  The  method  is -as  follows:  Render 
100  c.c.  of  urine  slightly  alkaline  with  sodium  carbonate  and  evaporate 
nearly  to  dryness  on  a  water-bath.  Dissolve  the  residue  in  150-200 
c.c.  of  50-55  per  cent  sulphuric  acid,  transfer  the  acid  solution  to  a 
i-liter  distillation  flask  and  connect  it  with  a  condenser.  Through 
the  cork  of  the  flask  introduce  the  stem  of  a  dropping  funnel  contain- 
ing water.  Heat  the  flask  gently  until  foaming  ceases,  then  use  a  full 
flame  and  distil  over  about  300-350  c.c.  of  fluid,  keeping  the  volume 
of  liquid  in  the  distillation  flask  constant  by  the  addition  of  water  from 
the  dropping  funnel  as  the  distillate  collects.  Ordinarily  it  will  take 
about  2-2  1/2  hours  to  collect  this  amount  of  distillate.  Extract  the 
distillate  three  times^  with  ether  in  a  separatory  funnel,  evaporate 
the  ether  and  heat  the  residue  at  160°  C.  for  a  few  minutes  to  remove 
volatile  fatty  acids.  Dissolve  the  residue  in  50  c.c.  of  water,  filter  and 
titrate  this  aqueous  solution  of  crotonic  acid  with  N/ 10  sodium  hydroxide 
solution,  using  phenolphthalein  as  indicator. 

Calculation. — One  c.c.  of  N/io  sodium  hydroxide  solution  equals 
0.0086  gram  of  crotonic  acid,  i  part  of  crotonic  acid  ec^uals  1.21 
part  of  i^-oxybutyric  acid,  and  i  c.c.  of  N/io  sodium  hydroxide  solu- 
tions equals  0.01041  gram  of  /3-oxybutyric  acid.  To  compute  the 
quantity  of  /5-oxybutyric  acid,  in  grams,  multiply  the  number  of 
cubic  centimeters  of  N/io  sodium  hydroxide  solution  used  by 
0.01041. 

4.  Bergell's  Method. — Render  100-300  c.c.  of  sugar-free-  urine 
slightly  alkaline  with  sodium  carbonate,  evaporate  the  alkaline  urine 
to  a  syrup  on  a  water-bath,  cool  the  syrup,  rub  it  up  with  syrupy 
phosphoric  acid  (being  careful  to  keep  the  mixture  cool),  20-30  grams 
of  finely  pulverized,  anhydrous  cupric  sulphate,  and  20-25  grams  of 
fine  sand.  Mix  the  mass  thoroughly,  place  it  in  a  paper  extraction 
thimble^  and  extract  the  dry  mixture  with  ether  in  a  Soxhlet  apparatus 
(Fig.  126,  page  405).  Evaporate  the  ether,  dissolve  the  residue  in 
about  25  c.c.  of  water,  decolorize  the  fluid  with  animal  charcoal,  if 

'  Shaffer  has  recently  called  attention  to  the  fact  that  it  is  extremely  difficult  to  extract 
all  of  the  crotonic  acid  if  but  three  extractions  are  made.  i 

^  If  sugar  is  present  it  must  be  removed  by  fermentation. 
^  The  Schleicher  and  Schiill  fat-free  extraction  thimble  is  very  satisfactory. 


398  PHYSIOLOGICAL    CHEMISTRY. 

necessary,  and  determine  the  content  of  .5-oxybulyric  acid  by  a  polariza- 
tion test. 

5.  Boekelman  and  Bouma's  Method. — Place  25  c.c.  of  urine  in 
a  flask,  add  25  c.c.  of  12  per  cent  sodium  hydroxide  and  25  c.c.  of 
benzoyl  chloride,  stopper  the  flask  and  shake  it  Aigorously  for  three 
minutes  under  cold  water.  Remove  the  clear  fluid  by  means  of  a 
pipette,  filter  it  and  subject  it  to  a  polarization  test.  Through  the 
action  of  the  benzoyl  chloride  all  the  tevo-rotatory  substances  ex- 
cept _.9-oxybutyric  acid  will  have  been  remo\cd  and  the  laevo-rotation 
now  exhibited  by  the  urine  will  be  due  entirely  to  that  acid. 

XVIII.  Acidity. 

Folin's  Method. — The  total  acidity  of  urine  may  be  determined 
as  follows:  Place  25  c.c.  of  urine  in  a  200  c.c.  Erlenmeyer  flask  and 
add  15-20  grams  of  finely  pulverized  potassium  oxalate  and  1-2  drops 
of  a  I  per  cent  phenolphthalein  solution  to  the  fluid.  Shake  the  mix- 
ture vigorously  for  1-2  minutes  and  titrate  it  immediately  with  N/io 
sodium  hydroxide  until  a  faint  but  unmistakable  pink  remains  perma- 
nent on  further  shaking.  Take  the  burette  reading  and  calculate  the 
acidity  of  the  urine  under  examination. 

Calculation. — If  y  represents  the  number  of  cubic  centimeters  of 
X/io  sodium  hydroxide  used  and  y'  represents  the  volume  of  urine 
excreted  in  twenty-four  hours,  the  total  acidity  of  the  twenty-four-hour 
urine  specimen  may  be  calculated  by  means  of  the  following  proportion: 

2-:,:y::y':x  (acidity  of  24-hour  urine  expressed  in  cubic  centimeters 
of  N/io  sodium  hydroxide). 

Each  cubic  centimeter  of  N/io  sodium  hydroxide  contains  0.004 
gram  of  sodium  hydroxide,  and  this  is  c(|ui\alenl  to  0.0063  gram  of 
oxalic  acid.  Therefore,  in  order  to  express  the  total  acidity  of  the 
twenty-four-hour  urine  specimen  in  equivalent  grams  of  sodium 
hydroxide,  multiply  the  value  of  x,  as  just  determined,  by  0.004, 
or  multijjly  the  value  of  x  by  0.0063  if  it  is  desired  to  express  the  total 
acidity  in  grams  of  oxalic  acid. 

XIX.     Purine  Bases. 

I .  Welker's  Modification  of  the  Methods  of  Arnstein  and  of 
Salkowski.'  Frmr  hundred  cubic  centimeters  of  urine,  free  from 
protein,  are  treated  with    roo  c.c.  of  magnesia   mixture  anrl  600  c.c. 

'  Private  communication  from  I)r.  W.  If.  Welkcr. 


urine:  quantitative  analysis.  399 

of  water.  This  is  then  filtered  and  of  the  clear  filtrate  a  measured 
quantity  (600-800  c.c.)  is  treated  with  an  excess  (10  c.c.)  of  a  3  per  cent 
silver  nitrate  solution.  Concentrated  ammonium  hydroxide  is  added 
in  small  quantities,  with  stirring,  until  all  the  chlorides  have  dissolved. 
Allow  the  flocculent  precipitate  of  the  silver  purine  compounds  to  settle 
to  the  bottom,  then  pass  the  supernatant  liquid  through  the  filter  before 
disturbing  the  precipitate.  Finally  transfer  the  precipitate  quantita- 
tively to  the  paper  which  must  be  of  known  nitrogen  content.  The 
precipitate  is  washed  with  dilute  (i  per  cent)  ammonium  hydroxide. 
The  paper  with  the  precipitate  is  then  transferred  to  a  Kjeldahl  flask 
and  about  100  c.c.  of  water  and  a  small  quantity  (about  o.i  gram)  of 
magnesium  oxide  are  added.  The  water  is  then  boiled  until  all  the 
ammonia  has  been  driven  off.     Test  the  steam  with  litmus  paper. 

The  material  in  the  flask  is  then  digested  by  means  of  the  usual 
Kjeldahl  method  (see  p.  375).  The  digestion  must  be  watched  care- 
fully at  the  time  the  sulphuric  acid  reaches  sufficient  concentration  to 
affect  the  filter  paper,  inasmuch  as  the  SO2  produced  causes  consider- 
able frothing.  The  total  nitrogen  (purine  base,  uric  acid  and  filter- 
paper  nitrogen)  is  now  determined  in  the  usual  way  (see  Kjeldahl 
Method,  p.  375).  This  result  minus  the  uric  acid  and  filter-paper 
nitrogen  will  give  the  figure  for  the  purine-base  nitrogen. 

2.  Kriiger  and  Schmidt's  Method. — This  method  serves  for  the 
determination  of  both  uric  acid  and  the  purine  bases.  The  principle 
involved  is  the  precipitation  of  both  the  uric  acid  and  the  purine  bases 
in  combination  with  copper  oxide  and  the  subsequent  decomposition 
of  this  precipitate  by  means  of  sodium  sulphide.  The  uric  acid  is 
then  precipitated  by  means  of  hydrochloric  acid  and  the  purine  bases 
are  separated  from  the  filtrate  in  the  form  of  their  copper  or  silver  com- 
pounds. The  nitrogen  content  of  the  precipitates  of  uric  acid  and 
purine  bases  is  then  determined  by  means  of  the  Kjeldahl  method 
(see  p.  375)  and  the  corresponding  values  for  uric  acid  and  purine 
bases  calculated.  The  method  is  as  follows:  To  400  c.c.  of  albumin- 
free  urine^  in  a  liter  flask,^  add  24  grams  of  sodium  acetate,  40  c.c. 
of  a  solution  of  sodium  bisulphite^  and  heat  the  mixture  to  boiling. 
Add  40-80  c.c*  of  a  10  per  cent  solution  of  cupric  sulphate  and  main- 
tain the  temperature  of  the  mixture  at  the  boiling-point  for  at  least 

'  If  albumin  is  present,  the  urine  should  be  heated  to  boiling,  acidified  mth  acetic 
add,  and  filtered. 

-  The  total  volume  of  urine  for  the  twenty-four  hours  should  be  sufliciently  diluted 
with  water  to  make  the  total  volume  of  the  solution  1600-2000  c.c. 

^  A  solution  containing  50  grams  of  Kahlbaum's  commercial  sodium  bisulphite  in 
100  c.c.  of  water. 

*  The  exact  amount  depending  upon  the  content  of  the  purine  bases. 


400  PHYSIOLOGICAL    CHEMISTRY. 

three  minutes.  Filter  off  the  flocculent  precipitate,  wash  it  with  hot 
water  until  the  wash  water  is  colorless,  and  return  the  washed  precipi- 
tate to  the  flask  by  puncturing  the  tip  of  the  filter  paper  and  washing 
the  precipitate  through  by  means  of  hot  water.  Add  water  until  the 
volume  in  the  flask  is  approximately  200  c.c,  heat  the  mixture  to 
boiling  and  decompose  the  precipitate  of  copper  oxide  by  the  addition 
of  30  c.c.  of  sodium  sulphide  solution.^  After  decomposition  is  com- 
plete, the  mixture  should  be  acidified  with  acetic  acid  and  heated  to 
boiling  until  the  separating  sulphur  collects  in  a  mass.  Filter  the  hot 
fluid  by  means  of  a  filter-pump,  wash  with  Jiol  water,  add  10  c.c.  of  10 
per  cent  hydrochloric  acid  and  evaporate  the  filtrate  in  a  porcelain 
dish  until  the  total  volume  has  been  reduced  to  about  10  c.c. 
Permit  this  residue  to  stand  about  two  hours  to  allow  for  the  sepa- 
ration of  the  uric  acid,  leaving  the  purine  bases  in  solution.  Filter 
off  the  precipitate  of  uric  acid,  using  a  small  filter  paper,  and  wash  the 
uric  acid,  with  water  made  acid  with  sulphuric  acid,  until  the  total 
volume  of  the  original  filtrate  and  the  wash  water  aggregates  75  c.c. 
Determine  the  nitrogen  content  of  the  precipitate  by  means  of 
the  Kjeldahl  method  (see  p.  375),  and  calculate  the  uric  acid 
equivalent.^ 

Render  the  filtrate  from  the  uric  acid  crystals  alkaline  with  sodium 
hydroxide,  add  acetic  acid  until  faintly  acid  and  heat  to  70°  C.  Now 
add  I  c.c.  of  a  10  per  cent  solution  of  acetic  acid  and  10  c.c.  of  a 
suspension  of  manganese  dioxide^  to  oxidize  the  traces  of  uric  acid 
which  remain  in  the  solution.  Agitate  the  mixture  for  one  minute, 
add  10  c.c.  of  the  sodium  bisulphite  solution"*  and  5  c.c.  of  a  10  per 
cent  solution  of  cupric  sulphate  and  heat  the  mixture  to  boiling  for 
three  minutes.  Fiher  off  the  j)reci|jitale,  wash  il  with  hoi  water, 
and  determine  its  nitrogen  c(jntent  by  means  of  the  Kjeldahl  method 
(see  p.  375).  Inasmuch  as  the  comj)osilion  and  projjortion  of  the 
purine  bases  present  in  urine  is  variable,  no  factor  can  be  applied. 
The  result  as  regards  these  bases  musl  ihcreforc  be  ex])ressed  in  terms 
of  nitrogen. 

'  'i'his  is  made  by  saturating  a  i  per  cent  solution  of  sodium  liydroxide  with  hydrogen 
sulphide  gas  anrl  adding  an  e(|ual  volume  of  i  per  cent  sodium  hydroxide. 

Ordinarily  the  adflition  of  30  f:.c.  of  this  solution  is  suOicicnl,  hul  the  presence  of  an 
excess  f>f  sulj;hidc  should  \)v  proven  hy  adrling  a  drojx)!  lead  acetate  lo  a  drop  of  the  solution. 
Under  these  conditions  a  dark  brown  (olor  will  show  the  |)resiiue  of  an  excess  of 
sodium  sulphide. 

'■'This  may  be  done  by  multiplying  the  nitrogen  value  by  three  and  adding  three  and 
f)ne-half  milligrams  to  the  product  as  a  correc  tion  for  the  urii  acid  remaining  in  solution 
in  the  75  c.c. 

'Made  by  heating  a  0.5  per  cent  solution  of  jjotassium  |)iriiiangaiiate  with  a  little- 
alcohol  until  it  is  riccolori/.ed. 

*  To  dissolve  the  exc  ess  of  manganese  dioxirle. 


urine:  quantitative  analysis.  4PI 

Benedict  and  Saiki^  report  cases  in  which  the  total  purine  nitrogen 
by  this  method  was  less  than  the  uric-acid  nitrogen  as  determined  by 
the  Folin-Shaffer  method.  The  inaccuracy  was  found  to  He  in  the 
Kriiger  and  Schmidt  method.  To  obviate  this  they  advise  the  addition 
of  20  c.c.  of  glacial  acetic  acid  for  each  300  c.c.  of  urine  employed^  the 
acid  being  added  before  the  first  precipitation. 

3.  Salkowski's  Method. — Place  400-600  c.c.  of  protein-free  urine 
in  a  beaker.  Introduce  into  another  beaker  30-50  c.c.  of  an  ammoni- 
acal  silver  solution^  with  30-50  c.c.  of  magnesia  mixture,^  add  some 
ammonium  hydroxide  and  if  necessary  some  ammonium  chloride  to 
clear  the  solution.  Now  add  this  solution  to  the  urine,  stirring  con- 
tinually with  a  glass  rod,  and  allow  the  mixture  to  stand  for  one-half 
hour.  Collect  the  precipitate  on  a  filter  paper,  wash  it  with  dilute 
ammonium  hydroxide,  and  finally  wash  it  back  into  the  original  beaker. 
Suspend  the  precipitate  in  600-800  c.c.  of  water,  add  a  few  drops  of 
hydrochloric  acid  and  decompose  it  by  means  of  hydrogen  sulphide. 
Now  heat  the  solution  to  boiling,  filter  while  hot  and  evaporate  the 
filtrate  to  dryness  on  a  water-bath.  Extract  the  residue  with  20-30  c.c. 
of  hot  3  per  cent  sulphuric  acid  and  allow  the  extract  to  stand  twenty- 
four  hours.  Filter  off  the  uric  acid,  wash  it,  make  the  filtrate  ammoni- 
acal  and  precipitate  the  purine  bases  again  with  silver  nitrate. 
Collect  this  precipitate  on  a  small-sized  chlorine-free  filter  paper,  wash, 
dry,  and  incinerate  it,  in  the  usual  manner.  Now  dissolve  the  ash  in 
nitric  acid  and  titrate  with  ammonium  thiocyanate  according  to  the 
Volhard- Arnold  method  (see  p.  390).  Calculate  the  content  of  purine 
bases  in  the  urine  examined,  bearing  in  mind  that  in  an  equal  mixture 
of  the  silver  salts  of  the  purine  bases,  such  as  we  have  here,  one  part 
of  silver  corresponds  to  0.277  gram  of  nitrogen  or  to  0.7381  gram  of 
the  bases. 

XX.    Allantoin. 

Paduschka-Underhill-Kleiner  Method. — To  50^-100  c.c.  of 
urine  in  a  beaker  add  basic  lead  acetate  until  no  more  precipitate 
forms.  Filter  and  pass  hydrogen  sulphide  gas  through  an  aliquot 
portion  of  the  filtrate  to  remove  the  excess  of  lead.*  Filter  again, 
drive  off  the  hydrogen  sulphide  by  heat  and  treat  an  aliquot  portion  of 

'  Benedict  and  Saiki:  Jotir.  Biol.  Chem.,  VII,  p.  27,  1909. 

-  Prepared  by  dissolving  26  grams  of  silver  nitrate  in  about  500  c.c.  of  water,  adding 
enough  ammonium  hydroxide  to  redissolve  the  precipitate  which  forms  upon  the  first 
addition  of  the  ammonia  and  malung  the  balance  of  the  mixture  up  to  i  liter  with  water. 

^  Directions  for  preparation  may  be  found  on  page  289. 

■'  In  the  original  method  of  Paduschka  sodium  sulphate  is  used  for  this  purpose. 
26 


402  PHYSIOLOGICAL    CHEMISTRY. 

•the  filtrate  with  a  lo  per  cent  solution  of  silver  nitrate  until  precipitation 
is  complete/  Filter  oft"  this  precipitate,  wash  it  with  water  and  deter- 
mine its  nitrogen  content  by  means  of  the  Kjeldahl  method  (see  p.  375). 
This  is  the  "purine  nitrogen."  Render  an  ahquot  portion  of  the 
fihrate  faintly  alkaline,^  with  a  i  per  cent  solution  of  ammonium 
hydroxide  and  add  50-100  c.c.  of  a  10  per  cent  solution  of  silver  nitrate. 
If  allantoin  be  present  a  white,  fiocculent  precipitate  will  form  and 
gradually  sink  to  the  bottom  of  the  solution.  Filter,  wash  the  precipi- 
tate free  from  ammonium  hydroxide  by  means  of  a  i  per  cent  solu- 
tion of  sodium  sulphate  and  determine  its  nitrogen  content  by  the 
Kjeldahl  method  (see  p.  375). 

XXI.     Oxalic  Acid. 

Salkowski-Autenrieth  and  Barth  Method. — Place  the  twenty- 
four-hour  urine  specimen  in  a  precipitating  jar,  add  an  excess  of 
calcium  chloride,  render  the  urine  strongly  ammoniacal,  stir  it  well, 
and  allow  it  to  stand  18-20  hours.  Filter  off  the  precipitate,  wash  it 
with  a  small  amount  of  water  and  dissolve  it  in  about  30  c.c.  of  a  hot 
15  per  cent  solution  of  hydrochloric  acid.  By  means  of  a  separatory 
funnel  extract  the  solution  with  150  c.c.  of  ether  which  contains  3  per 
cent  of  alcohol,  repeating  the  extraction  four  or  five  times  with  fresh 
portions  of  ether.  Unite  the  ethereal  extracts,  allow  them  to  stand 
for  an  hour  in  a  flask,  and  then  filter  through  a  dry  filter  paper.  Add 
5  c.c.  of  water  to  the  filtrate,  to  prevent  the  formation  of  diethyl  oxalate 
when  the  solution  is  heated,  and  distil  off  the  ether.  If  necessary, 
decolorize  the  li(|uid  with  animal  charcoal  and  filter.  Concentrate 
the  filtrate  to  3-5  c.c,  add  a  little  calcium  chloride  solution,  make  it 
ammoniacal.  and  after  a  few  minutes  render  it  slightly  acid  with  acetic 
acid.  Allow  the  acidified  solution  to  stand  se\eral  hours,  collect  the 
precipitate  of  calcium  oxalate  on  a  washed  filter  paper,^  wash,  inciner- 
ate strongly  (to  CaO),  and  weigh  in  the  usual  manner. 

Calculalion. -  ^'mcc  56  parts  of  CaO  are  e(|ui\'alen(  to  go  j)arts  of 
oxalic  acid,  the  (juantity  of  oxalic  acid  in  the  volume  of  urine  taken 
may  be  determined  by  multiplying  the  weight  of  CaO  by  the  factor 
[.6071. 

XXII.     Total  Solids. 

I.  Drying  Method.  Place  5  c.c.  of  mine  in  a  weighed  shallow 
dish,  acidify  very  slightly  willi  acclic  acid   fi    •;  drops),  and  flry  it   /// 

'  Ordinarily  from  20- .^o  c.r.  is  re(|uirc(l. 

-  Using  lilmus  as  the  inriicator. 

■'  Sfhlfii  her  and  Schull,  .No.  58(7,  is  satisfactory. 


urine:  quantitative  analysis.  403 

vacuo  in  the  presence  of  sulphuric  acid  to  constant  weight.  Calculate 
the  percentage  of  solids  in  the  urine  sample  and  the  total  solids  for  the 
twenty-four-hour  period. 

Practically  all  the  methods  the  technique  of  which  includes  evapo- 
ration at  an  increased  temperature,  either  under  atmospheric  conditions 
or  in  vacuo,  are  attended  with  error. 

2.  Calculation  by  Long's  Coefficient. — The  quantity  of  solid 
material  contained  in  the  urine  excreted  for  any  twenty-four-hour 
period  may  be  approximately  computed  by  multiplying  the  second  and 
third  decimal  figures  of  the  specific  gravity  by  2.6.  This  gives  us  the 
number  of  grams  of  solid  matter  in  i  liter  of  urine.  From  this  value 
the  total  solids  for  the  twenty-four-hour  period  may  easily  be 
determined. 

Calculation. — If  the  volume  of  urine  for  the  twenty-four  hours  was 
1120  c.c.  and  the  specific  gravity  1.018,  the  calculation  would  be  as 
follows : 

{a)         18X2.6  =  46.8  grams  of  sohd  matter  in  i  hter  of  urine. 

,,s    46.8X1120     ^  ,      ,.,        ^^      .  ... 

ib)   =  ^2  .4  grams  of  sohd  matter  m  11 20  c.c.  of  unne. 

^  1000  ^        ^ 

Long's  coefficient  was  determined  for  urine  whose  specific  gravity 
was  taken  at  25°  C.and  is  probably  more  accurate,  for  conditions 
obtaining  in  America,  than  the  older  coefficient  of  Haeser,  2.33. 


CHAPTER  XXIII. 


QUANTITATIVE  ANALYSIS  OF  MILK,  GASTRIC  JUICE,  AND 

BLOOD. 

(a)  Quantitative  Analysis  of  Milk. 

I.  Specific  Gravity.— This  may  be  determined  conveniently  by 
means  of  a  Soxhlet,  \'eith,  or  Quevenne  lactometer.  A  lactometer 
reading  of  32°  denotes  a  specific  gravity  of  1.032.     The  determination 

should  be  made  at  about  60°  F.  and  the 
lactometer  reading  corrected  by  adding 
or  subtracting  0.1°  for  every  degree  F. 
above  or  below  that  temperature. 

2.  Fat. — {a)  Quantitative  Deiermina- 
tion  of  Fat  in  Milk  by  the  Meigs^  Method 
with  Modification  and  Improved  Appa- 
ratus by  Croll.  - — The  method  as  stated  by 
Dr.  Meigs  is:  Approximately  10  c.c.  of 
milk  is  carefully  weighed  and  transferred 
to  an  ordinary  100  c.c.  glass-stoppered 
graduated  cylinder.  Twenty  c.c.  each 
of  distilled  water  and  ether  (0.720)  are 
added,  the  ground-glass  stopper  tightly 
inserted  in  the  bottle,  and  the  whole 
shaken  vigorously  for  five  minutes. 
Then  the  bottle  is  carefully  unstoppered, 
20  c.c.  95  per  cent  alcohol  added,  the 
stopper  reinserted  and  again  shaken  for 
five  minutes.  The  bottle  is  now  placed 
on  a  table  and  the  contents  will  separate 
into  two  distinct  strata,  the  upper  of 
which  contains  ])ra(tically  all  the  fat. 
This  stratum  is  carefully  removed  by  a 
small  pipette  and  transferred  to  a  care- 
fully weighed  glass  evaporating  dish. 
The  thin  ether  layer  remaining  is  washed  by  ihc  addition  of  5  c.c.  of 
ether.     This  is  removed  by  pipetting  off.     This  washing  is  repeated 

paper     by     Dr.    Arthur   V.    Mi-if^s    in     I'liilndrljiliia     Medical    Times, 


J-k;.  iJv     <  KOI. IS  Iai  An-AKATUS. 


'  Original 
July   r,  i««2 

^  Private  Communitation. 


404 


QUANTITATIVE   ANALYSIS    OF    MILK. 


405 


four  times.  On  each  addition  the  sides  of  the  bottle  should  carefully 
be  washed  down  by  the  fresh  ether.  Finally,  the  pipette  is  rinsed 
with  a  little  ether.  The  evaporating  dish  with  contents  is  now  placed 
on  a  safety  water-bath  and  the  ether  evaporated.  The  drying  is  con- 
tinued in  a  hot-air  oven  at  a  temperature  below  100°  C.  and  finally 
completed  in  a  desiccator  to  constant 
weight. 

Croll's  modification  consists  of  sub- 
sequent repeated  extraction  of  the  end- 
product  of  evaporation  with  absolute 
ether.  The  combined  extracts  are 
filtered  and  the  small  filter  paper  is 
washed  repeatedly  with  absolute  ether. 
The  combined  extracts  and  washings 
are  evaporated  and  dried  as  before  and 
then  weighed. 

The  piece  of  apparatus  shown  in 
Fig.  125,  p.  404  was  also  devised  by  Croll 
to  do  away  with  the  use  of  the  pipette. 
On  closing  the  top  with  a  finger  and 
blowing  into  the  mouth-piece,  the  upper 
stratum  is  forced  out  into  the  dish. 
The  bottle  is  washed  by  simply  pouring 
the  ether  into  the  tube.  This  lessens 
the  possibility  of  accidental  loss. 

The  accuracy  of  the  method  com- 
pared with  that  of  the  Soxhlet  method, 
using  the  paper-coil  modification  and 
extracting'  until  fresh  portions  of  abso- 
lute ether  gave  no  further  trace  of  ex- 
tractive material,  is  shown  by  the 
average  difference  on  twelve  samples  of 

human  milk  being  only  0.017  P^^  cent  less  than  by  the  Soxhlet  and 
on  seven  samples  cow's  milk  being  only  0.019  per  cent  less.  The 
extreme  differences  in  case  of  the  human  milk  were  —0.004  per  cent 
and— 0.044  per  cent  and  in  case  of  the  cow's  milk— 0.006  per  cent  and 
—0.068  per  cent. 

(b)  Adams'  Paper-coil  Method. — Introduce  about  5  c.c.  of  milk 
into  a  small  beaker,  quickly  ascertain  the  weight  to  centigrams,  stand 
a  fat-free  coiP  in  the  beaker,  and  incline  the  vessel  and  rotate  the  coil 


Fig.  126. — Soxhlet  .\ppar.\tus. 


'Very  satisfactor}'  coils  are  manufactured  by  Schleicher  and  Schlill. 


4o6 


PHYSIOLOGICAL    CHEMISTRY. 


in  order  to  hasten  the  absorption  of  the  milk.  Immediately  upon  the 
complete  absorption  of  the  milk  remove  the  coil  and  again  quickly 
ascertain  the  weight  of  the  beaker.  The  difference  in  the  weights  of 
the  beaker  at  the  two  weighings  represents  the  quantity  of  milk  absorbed 
by  the  coil.  Dry  the  coil  carefully  at  a  temperature  Ijelow  ioo°  C. 
and  extract  it  with  ether  for  3-5  hours  in  a  Soxhlet  apparatus  (Fig.  126, 
p.  405).  Using  a  safety  water-bath,  heat  the  flask  containing  the  fat 
to  constant  weight  at  a  temperature  below  100°  C. 

Calculatian. — Divide  the  weight  of  fat,  in  grams,  by  the  weight  of 
milk,  in  grams.  The  quotient  is  the  perrcnla^i^e  of  fat  contained  in  the 
milk  examined. 

(f)  Approximate  Determination  by  Feser\s  Lactoscope. — Milk  is 
opaque  mainly  because  of  the  suspended  fat  globules  and  therefore 
by  means  of  the  estimation  of  this  opacity  we  may 
obtain  data  as  to  the  approximate  content  of  fat. 
Feser's  lactoscope  (Fig.  127)  may  be  used  for  this 
purpose.  Proceed  as  follows:  By  means  of  the 
graduated  pipette  accompanying  the  instrument  in- 
troduce 4  c.c.  of  milk  into  the  lactoscope.  Add 
water  gradually,  shaking  after  each  addition,  and  note 
the  point  at  which  the  black  lines  upon  the  inner 
white  glass  cylinder  are  distinctly  visible.  Observe 
the  point  on  the  graduated  scale  of  the  lactoscope 
which  is  le\'el  with  the  surface  of  the  diluted  milk. 
This  reading  represents  the  percentage  of  fat  present 
in  the  undiluted  milk.  Pure  milk  should  contain  at  Fig.  127. —Feser's 
least  3  per  cent  of  fat.  Lactoscope. 

3.  Total  Solids.^ — Introduce  2-5  grams  of  milk  into  a  weighed 
flat-bottomed  platinum  dish^  and  quickly  ascertain  the  weight  to 
milligrams.  IOxjk-1  the  major  portion  of  the  water  by  heating  the  open 
dish  on  a  water  balh  and  continue  the  heating  in  an  air-bath  or  water 
oven  at  97°-ioo°  C.  until  the  weight  is  constant.  (If  platinum  dishes 
are  employed  this  residue  may  be  used  in  the  determination  of  asli 
according  to  the  method  described  on  ]>.  407.) 

Calculatinii.      I)i\ir|c   ihc   weight   of  the  residue,   in   grams,  by  the 

'  'rill-  (jCTcentanc;  <>i  lota!  solids  may  tjc  calculated  from  llic  specific  gravity  and  per- 
centage of  fat  by  means  of  the  following  formula  wlii(  li  lias  been  i)roj)osed  by  Richmond: 

S  =  o .  25  L  -I- 1 . 2  F  +  o .  1 4 
S  =  total  solids. 
I/"=  lactometer  reading. 
F  —  fat  content. 

*  Lead  foil  riishes,  costing  f)nly  aboui  one  dollar  jjcr  gross,  make  a  very  satisfa(  tor\' 
substitute  for  the  platinum  tlishcs. 


QUANTITATIVE   ANALYSIS    OF    MILK.  407 

weight  of  milk  used,  in  grams.    The  quotient  is  the  percentage  of  solids 
contained  in  the  milk  examined. 

4.  Ash. — Heat  the  dry  solids  from  2-5  grams  of  milk,  obtained 
according  to  the  method  just  given,  over  a  very  low  flame'-  until  a 
white  or  light  gray  ash  is  obtained.  Cool  the  dish  in  a  desiccator  and 
weigh.  (This  ash  may  be  used  in  testing  for  preservatives  according 
to  directions  on  page  221. 

5.  Proteins. — Introduce  a  known  weight  of  milk  (5-10  grams) 
into  a  500  c.c.  Kjeldahl  digestion  flask  and  add  20  c.c.  of  concentrated 
sulphuric  acid  and  about  0.2  gram  of  cupric  sulphate.  Expel  the  major 
portion  of  the  water  by  heating  over  a  low  flame  and  finally  use  a  full 
flame  and  allow  the  mixture  to  boil  1-2  hours.  Complete  the  deter- 
mination according  to  the  directions  given  under  Kjeldahl  Method^ 

page  375- 

Calculation. — Multiply  the  total  nitrogen  content  by  the  factor  6.37^ 
to  obtain  the  protein  content  of  the  milk  examined. 

6.  Caseinogen. — Mix  about  20  grams  of  milk  with  40  c.c.  of  a 
saturated  solution  of  magnesium  sulphate  and  add  the  salt  in  sub- 
stance until  no  more  will  dissolve.  The  precipitate  consists  of  Qasein- 
ogen  admixed  with  a  little  fat  and  lacto-globulin.  Filter  off  the  pre- 
cipitate, wash  it  thoroughly  with  a  saturated  solution  of  magnesium 
sulphate,^  transfer  the  filter  paper  and  precipitate  to  a  Kjeldahl  diges- 
tion flask,  and  determine  the  nitrogen  content  according  to  the  direc- 
tions given  in  the  previous  experiment. 

Calculation. — Multiply  the  total  nitrogen  by  the  factor  6.37  to 
obtain  the  casein  content. 

7.  Lactalbumin. — To  the  filtrate  and  washings  from  the  deter- 
mination of  caseinogen,  as  just  explained,  add  Almen's  reagent^  until 
no  more  precipitate  forms.  Filter  off  the  precipitate .  and  determine 
the  nitrogen  content  according  to  the  directions  given  under  Proteins, 
above. 

Calculation. — Multiply  the  total  nitrogen  by  the  factor  6.37  to 
obtain  the  lactalbumin  content. 

8.  Lactose. — To  about  350  c.c.  of  water  in  a  beaker  add  20  grams 

^  Great  care  should  be  used  in  this  ignition,  the  dish  at  no  time  being  heated  above  a 
faint  redness,  as  chlorides  may  volatilize. 

"  The  usual  factor  employed  for  the  calculation  of  protein  from  the  nitrogen  content 
is  6.25  and  is  based  on  the  assumption  that  proteins  contain  on  the  average  i6  per  cent 
of  nitrogen.  This  special  factor  of  6.37  is  used  here  to  calculate  the  protein  content  from 
the  total  nitrogen,  since  the  principal  protein  constituents  of  milk,  i.  e.,  caseinogen  and 
lactalbumin,  contain  15  .7  per  cent  of  nitrogen. 

'  Preserve  the  filtrate  and  washings  for  the  determination  of  lactalbumin. 

*  Almen's  reagent  may  be  prepared  by  dissolving  5  grams  of  tannin  in  240  c.c.  of  50 
per  cent  alcohol  and  adding  10  c.c.  of  25  per  cent  acetic  acid. 


4o8  PHYSIOLOGICAL    CHEMISTRY. 

of  milk,  mix  thoroughly,  acidify  the  fluid  with  about  2  c.c.  of  10  per 
cent  acetic  acid  and  stir  the  acidified  mixture  continuously  until  a 
flocculent  precipitate  forms.  At  this  point  the  reaction  should  be 
distinctly  acid  to  litmus.  Heat  the  solution  to  boiling  for  one-half 
hour,  filter,  rinse  the  beaker  thoroughly,  and  wash  the  precipitated 
proteins  and  the  adherent  fat  with  hot  water.  Combine  the  filtrate 
and  wash  water  and  concentrate  the  mixture  to  about  150  c.c.  Cool 
the  solution  and  dilute  it  to  200  c.c.  in  a  volumetric  flask.  Titrate  this 
sugar  solution  according  to  directions  given  under  Fehling's  Method, 
page  362. 

Calculation. — Make  the  calculation  according  to  directions  given 
under  Fehling's  Method,  p.  362,  bearing  in  mind  that  10  c.c.  of 
Fehling's  solution  is  completely  reduced  by  0.0676  gram  of  lactose. 

(b)  Quantitative  Analysis  of  Gastric  Juice. 

Topfer's  Method. 

This  method  is  much  less  elaborate  than  many  others  Init  is  sufti- 
ciently  accurate  for  ordinary  clinical  purposes.  The  method  embraces 
the  volumetric  determination  of  (i)  total  acidity,  (2)  combined  acidity, 
and  (3)  free  acidity,  and  the  subsequent  calculation  of  (4)  acidity  due 
to  organic  acids  and  acid  salts,  from  the  data  thus  obtained. 

Strain  the  gastric  contents  and  introduce  10  c.c.  of  the  strained 
material  into  each  of  three  small  beakers  or  porcelain  dishes.'  Label 
the  vessels  A,  B  and  C,  respectively,  and  proceed  with  the  analysis 
according  to  the  directions  given  below. 

I.  Total  Acidity." — Add  3  drops  of  a  i  per  cent  alcoholic  solution 
of  phenolphthalein''  to  the  contents  of  vessel  A  and  titrate  with  N/io 
sodium  hydroxide  solution  until  a  dark  pink  color  is  produced  which 
cannot  be  deepened  by  further  addition  of  a  drop  of  N/io  sodium 
hydroxide.    Take  the  burette  reading  and  calculate  the  total  acidity. 

Calculation. — The  total  acidity  may  be  expressed  in  the  following 
ways: 

1.  The  number  (jf  cubic  centimeters  of  N/io  sodium  hydroxide 
solution  necessary  to  neutralize  too  c.c.  of  gastric  juice. 

2.  The  weight  (in  grams)  of  sodium  hydroxide  necessary  to  neu- 
tralize 100  c.c.  of  gastric  juice. 

*  If  suflTicient  gastric  juice  is  not  av;iiia)>le  it  may  he  diluted  witli  \v;itcr  or  a  smaller 
amoOnt,  e.  g.,  5  c.c.  taken  for  each  determination. 

-  This  inclurles  free  an<l  comhincd  acid  and  acid  salts. 

■'  One  gram  of  jihenolphthalein  dissolved  in  loo  c.c.  of  95  per  (  ent  ali ohol. 


QUANTITATIVE  ANALYSIS    OF    GASTRIC   JUICE.  409 

3.  The  weight  (in  grams)  of  hydrochloric  acid  which  the  total 
acidity  of  loo  c.c.  of  gastric  juice  represents,  i.  e.,  percentage  of  hydro- 
chloric acid. 

The  forms  of  expression  most  frequently  employed  are  i  and  3, 
preference  being  gi\-en  to  the  former. 

In  making  the  calculation  note  the  number  of  cubic  centimeters  of 
N/io  sodium  hydroxide  required  to  neutralize  10  c.c.  of  the  gastric 
juice  and  multiply  it  by  10  to  obtain  the  number  of  cubic  centimeters 
necessary  to  neutralize  100  c.c.  of  the  fluid.  If  it  is  desired  to  express 
the  acidity  of  100  c.c.  of  gastric  juice  in  terms  of  hydrochloric  acid, 
by  weight,  multiply  the  value  just  obtained  by  0.00365.^ 

2.  Combined  Acidity.^ — Add  3  drops  of  sodium  ahzarin  sulpho- 
nate  solution^  to  the  contents  of  vessel  B  and  titrate  with  N/io  sodium 
hydroxide  solution  until  a  violet  color  is  produced.  In  this  titration 
the  red  color,  which  appears  after  the  tinge  of  yellow  due  to  the  addi- 
tion of  the  indicator  has  disappeared,  must  be  entirely  replaced  by  a 
distinct  violet  color.  Take  the  burette  reading  and  calculate  the  com- 
bined  acidity. 

Calculation. — Since  the  indicator  used  reacts  to  all  acidities  except 
combined  acidity,  in  order  to  determine  the  number  of  cubic  centimeters 
of  N/io  sodium  hydroxide  necessary  to  neutralize  the  combined  acidity 
of  10  c.c.  of  the  gastric  juice,  we  must  subtract  the  burette  reading 
just  obtained  from  the  burette  reading  obtained  in  the  determination 
of  the  total  acidity.  The  data  for  100  c.c.  of  gastric  juice  may  be  cal- 
culated according  to  the  directions  given  under  Total  Acidity, 
page  408. 

3.  Free  Acidity/ — Add  4  drops  of  di-methyl-amino-azobenzene 
(Topfer's  reagent)  solution^  to  the  contents  of  the  vessel  C  and  titrate 
with  N/io  sodium  hydroxide  solution  until  the  initial  red  color  is  re- 
placed by  lemon  yellow.^  Take  the  burette  reading  and  calculate  the 
free  acidity. 

Calculation. — The  indicator  used  reacts  only  to  free  acid,  hence 
the  number  of  cubic  centimeters  of  N/ 10  sodium  hydroxide  used  in- 
dicates the  volume  necessary  to  neutralize  the  free  acidity  of  10  c.c.  of 
gastric  juice.  To  determine  the  data  for  100  c.c.  of  gastric  juice 
proceed  according  to  the  directions  given  under  Total  Acidity,  page  408. 

'  One  c.c.  of  N/io  hydrochloric  acid  contains  0.00365  gram  of  hydrochloric  acid. 
-  Hydrochloric  acid  combined  with  protein  material. 
^  One  gram  of  sodium  alizarin  sulphonate  dissolved  in  100  c.c.  of  water. 
^  Hydrochloric  acid  not  combined  with  protein  material. 
'  One-half  gram  dissolved  in  100  c.c.  of  95  per  cent  alcohol. 

**  If  the  lemon  yellow  color  appears  as  soon  as  the  indicator  is  added  it  denotes  the 
absence  of  free  acid. 


4IO  PHYSIOLOGICAL    CHEMISTRY. 

4.  Acidity  Due  to  Organic  Acids  and  Acid  Salts. — This  value 
may  be  convenienlly  calculated  by  subtracting  the  number  of  cubic 
centimeters  of  N/io  sodium  hydroxide  used  in  neutralizing  the  con- 
tents of  vessel  C  from  the  number  of  cubic  centimeters  of  N/io  sodium 
hydroxide  solution  used  in  neutralizing  the  contents  of  vessel  B.  The 
remainder  indicates  the  number  of  cubic  centimeters  of  N/io  sodium 
hydroxide  solution  necessary  to  neutralize  the  acidity  due  to  organic 
acids  and  acid  salts  present  in  10  c.c.  of  gastric  juice.  The  data  for 
100  c.c.  of  gastric  juice  may  be  calculated  according  to  directions 
given  under  Total  Acidity,  page  408. 

(c)  Quantitative  Analysis  of  Blood. 

For  the  methods  involved  in  the  quantitative  examination  of  blood 
see  Chapter  XII. 


APPENDIX. 

Almen's  Reagent/ — Dissolve  5  grams  of  tannin  in  240  c.c.  of 
50  per  cent  alcohol  and  add  10  c.c.  of  25  per  cent  acetic  acid. 

Ammoniacal  Silver  Solution.^ — Dissolve  26  grams  of  silver 
nitrate  in  about  500  c.c.  of  water,  add  enough  ammonium  hydroxide 
to  redissolve  the  precipitate  which  forms  upon  the  first  addition  of  the 
ammonium  hydroxide  and  make  the  volume  of  the  mixture  up  to  i 
liter  with  water. 

Arnold-Lipliawsky  Reagent.^ — This  reagent  consists  of  two 
definite  solutions  which  are  ordinarily  preserved  separately  and  mixed 
just  before  using.    The  two  solutions  are  prepared  as  follows: 

(a)  One  per  cent  aqueous  solution  of  potassium  nitrite. 

(b)  One  gram  of  />-amino-acetophenon  dissolved  in  100  c.c.  of 
distilled  water  and  enough  hydrochloric  acid  (about  2.  c.c)  added 
drop  by  drop,  to  cause  the  solution,  which  is  at  first  yellow,  to  become 
entirely  colorless.    An  excess  of  acid  must  be  avoided. 

Barfoed's  Solution/ — Dissolve  4.5  grams  of  neutral,  crystallized 
cupric  acetate  in  100  c.c.  of  water  and  add  1.2  c.c.  of  50  per  cent 
acetic  acid. 

Baryta  Mixture/ — A  mixture  consisting  of  one  volume  of  a 
saturated  solution  of  barium  nitrate  and  two  volumes  of  a  saturated 
solution  of  barium  hydroxide. 

Benedict's  Solutions/ — First  Modification. — Benedict's  modi- 
fied Fehling  solution  consists  of  two  definite  solutions — a  cupric  sul- 
phate solution  and  an  alkaline  tartrate  solution,  which  may  be  prepared 
as  follows: 

Cupric  sulphate  solution  =^1,4.6^  grams  of  cupric  sulphate  dissolved 
in  water  and  made  up  to  500  c.c. 

Alkaline  tartrate  solution  =  100  grams  of  anhydrous  sodium  car- 
bonate and  173  grams  of  Rochelle  salt  dissolved  in  water  and  made  up 
to  TOO  c.c. 

'  Ott's  precipitation  test,  p.  315.     Determination  of  lactalbumin,  p.  407.  , 

-  Salkowski's  method,  page  401. 
^  Amold-Lipliawsky  reaction,  page  326. 
^  Barfoed's  test,  pages  30  and  307. 
•'  Isolation  of  urea  from  urine,  page  263. 

®  Benedict's  modifications  of  Fehling's  test,  pages  27  and  303,  and  Benedict's  ^lethod. 
page  363. 

411 


412  PHYSIOLOGICAL    CHEMISTRY. 

These  solutions  should  be  preserved  separately  in  rubber-stoppered 
bottles  and  mixed  in  equal  volumes  when  needed  for  use.  This  is 
done  to  prevent  deterioration. 

Secand  Modification. — Very  recently  Benedict  has  further  modi- 
fied his  solution  and  has  succeeded  in  obtaining  one  which  does  not 
deteriorate  upon  long  standing.     It  has  the  following  composition: 

Cupric  sulphate    17.3  grams. 

Sodium  citrate    i73  -o  grams. 

Sodium  carbonate    100 .0  grams. 

Distilled  water  to  make  i  liter. 

With  the  aid  of  heat  dissolve  the  sodium  citrate  and  carbonate  in 
about  600  c.c.  of  water.  Pour  (through  a  folded  filter  paper  if  neces- 
sary) into  a  glass  graduate  and  make  up  to  850  c.c.  Dissolve  the  cupric 
sulphate  in  about  100  c.c.  of  water  and  make  up  to  150  c.c.  Pour  the 
carbonate-citrate  solution  into  a  large  beaker  or  casserole  and  add  the 
cupric  sulphate  solution  slowly,  with  constant  stirring.  The  mixed 
solution  is  ready  for  use  and  does  not  deteriorate  upon  long  standing. 

Benedict's  solution  as  used  in  the  quantitative  determination  of  sugar 
consists  of  three  separate  solutions,  the  two  mentioned  under  First 
Modification  and  in  addition  a  potassium  ferro-thiocyanate  solution.  This 
third  solution  contains  15  grams  of  potassium  ferrocyanide,  62.5 
grams  of  potassium  thiocyanate  and  50  grams  of  anhydrous  sodium 
carbonate  dissolved  in  water  and  made  up  to  500  c.c.  In  preparing 
the  Benedict's  solution  for  quantitative  work  the  tlirce  solutions  men- 
tioned are  combined  in  equal  parts. 

Benedict's  Sulphur  Reagent. 

Sodium  or  potassium  chlorate 50  grams. 

Distilled  water  to 1000  c.c. 

Crystallized  copper  nitrate,   sulphur-free  or    of    known 

sulphur  content 200  grams. 

Black's  Reagent.^ — Made  by  dissolving  5  grams  of  ferric  chloride 
and  0.4  gram  of  ferrous  chloride  in  joo  c.c.  of  water. 

Boas'  Reagent.^ — Dissolve  5  grams  of  rcsorcin  and  3  grams  of 
sucrose  in  100  c.c.  of  95  per  cent  alcohol. 

Bonnano's  Reagent.  Dissolve  2  grams  of  sodium  nitrite  in  100 
c.c.  of  concentrated  hydrochloric  acid. 

3ottu's  Reagent.  —To  3.5  grams  of  o-nitroi)hcnyl})roj)iolic  acid 
add  5  c.c.  of  a  freshly  prepared  10  per  cent  solution  of  sodium  hydroxide 
and  make  the  volume  of  the  solution  one  liter  with  distilled  water. 

'  Black's  reaction,  page  .327. 
"  Test  for  free  acid,  j^age  120, 


APPENDIX.  413 

.Congo  Red/ — Dissolve  0.5  gram  of  congo  red  in  90  c.c.  of  water 
and  add  10  c.c.  of  95  per  cent  alcohol. 

Cross  and  Bevan's  Reagent. — Combine  two  parts  of  concentrated 
hydrochloric  acid  and  one  part  of  zinc  chloride  by  weight. 

Ehrlich's  Diazo  Reagent.^ — Two  separate  solutions  should  be 
prepared  and  mixed  in  definite  proportions  when  needed  for  use. 

(a)  Five  grams  of  sodium  nitrite  dissolved  in  i  liter  of  distilled 
water. 

(&)  Five  grams  of  sulphanilic  acid  and  50  c.c.  of  hydrochloric  acid 
in  I  liter  of  distilled  water. 

Solutions  a  and  h  should  be  preserved  in  well-stoppered  vessels  and 
mixed  in  the  proportion  i  :  50  when  required.  Green  asserts  that 
greater  delicacy  is  secured  by  mixing  the  solutions  in  the  proportion 
I  :  100.  The  sodium  nitrite  deteriorates  upon  standing  and  becomes 
unfit  for  use  in  the  course  of  a  few  weeks. 

Esbach's  Reagent.^ — Dissolve  10  grams  of  picric  acid  and  20 
grams  of  citric  acid  in  i  liter  of  water. 

Fehling's  Solution/ — Fehling's  solution  is  composed  of  two 
definite  solutions — a  cupric  sulphate  solution  and  an  alkaline  tartrate 
solution,  which  may  be  prepared  as  follows: 

Cupric  sulphate  solution  =  ^4.6^  grams  of  cupric  sulphate  dissolved 
in  water  and  made  up  to  500  c.c. 

Alkaline  tartrate  solution=^i2^  grams  of  potassium  hydroxide  and 
173  grams  of  Rochelle  salt  dissolved  in  water  and  made  up  to 
500  c.c. 

These  solutions  should  be  preserved  separately  in  rubber-stoppered 
bottles  and  mixed  in  equal  volumes  when  needed  for  use.  This  is 
done  to  prevent  deterioration. 

Ferric  Alum  Solution/ — A  cold  saturated  solution. 

Folin-Shaffer  Reagent/ — This  reagent  consists  of  500  grams  of 
ammonium  sulphate,  5  grams  of  uranium  acetate,  and  60  c.c.  of  10 
per  cent  acetic  acid  in  650  c.c.  of  distilled  water. 

Furfurol  Solution/ — Add  i  c.c.  of  furfurol  to  1000  c.c.  of  distilled 
water. 

Gallic  Acid  Solution/ — A  saturated  alcoholic  solution. . 

'  Test  for  free  acid,  page  121. 
-  Ehrlich's  diazo  reaction,  page  336. 
^  Esbach's  method,  page  361. 

■*  Fehling's  method,  page  362.     Fehling's  test,  pages  27  and  303. 
°  Volhard-Arnold  method,  page  390. 
"Folin-Shaffer  method,  page  366. 

■^  Mylius's  modification  of  Pettenkofer's  test,  pages  153  and  320.  v.  Udransky's  test, 
pages  153  and  321. 

^  Gallic  acid  test,  page  220. 


414  PHYSIOLOGICAL    CHEMISTRY. 

Gies'  Biuret  Reagent. — Thisreagcnl  consists  of  lo  percent  KOH 
solution  to  which  enough  3  per  cent  CuSO^  solution  has  been  added 
to  impart  a  slight  though  distinct  blue  color  to  the  clear  liquid.  The 
CuSO^  should  be  added  drop  by  drop  with  thorough  shaking  after 
each  addition. 

Guaiac  Solution.^ — Dissolve  0.5  gram  of  guaiac  resin  in  30  c.c. 
of  95  per  cent  alcohol. 

Giinzberg's  Reagent." — Dissolve  2  grams  of  phloroglucin  and  i 
gram  of  \anillin  in  100  c.c.  of  95  per  cent  alcohol. 

Hammarsten's  Reagent.^ — Mix  i  volume  of  25  per  cent  nitric 
acid  and  19  volumes  of  25  per  cent  hydrochloric  acid  and  add  i  volume 
of  this  acid  mixture  to  4  volumes  of  95  per  cent  alcohol.  It  is  prefer- 
able that  the  acid  mixture  be  prepared  in  advance  and  allowed  to 
stand  until  yellow  in  color  before  adding  it  to  the  alcohol. 

Hopkins-Cole  Reagent. '^To  one  liter  of  a  saturated  solution  of 
oxalic  acid  add  Oo  grams  of  sodium  amalgam  and  allow  the  mixture 
to  stand  until  the  evolution  of  gas  ceases.  Filter  and  dilute  with  2-3 
\olumes  of  water. 

Hopkins-Cole  Reagent  (Benedict's  Modification). — Ten  grams 
of  powdered  magnesium  are  placed  in  a  large  Erlenmeyer  flask  and 
shaken  up  with  enough  distilled  water  to  liberally  cover  the  mag- 
nesium. Two  hundred  and  fifty  cubic  centimeters  of  a  cold,  saturated 
solution  of  oxalic  acid  is  now  added  slowly.  The  reaction  proceeds 
very  rapidly  and  with  the  liberation  of  much  heat,  so  that  the  flask 
should  be  cooled  under  running  water  during  the  addition  of  the  acid. 
The  contents  of  the  llask  are  shaken  after  the  addition  of  the  last  por- 
tion of  the  acid  and  then  poured  upon  a  filter,  to  remo\e  the  insoluble 
magnesium  oxalate.  A  little  wash  water  is  poured  through  the  filter, 
the  filtrate  acidified  with  acetic  acid  to  ]jre\'ent  the  partial  precipita- 
tion of  the  magnesium  on  long  standing,  and  made  up  to  a  liter  with 
distilled  water.  This  solution  contains  only  the  magnesium  salt  of 
glyoxylic  acid. 

Hypobromite  Solution/-  The  ingredients  of  this  solution  should 
be  prepared  in  the  form  of  tivo  separate  solutions  which  may  be  united 
as  needed. 

(a)  Dissoh'e  125  grams  of  sodium  l)romide  in  water,  add  125 
grams  of  bromine  and  make  the  toUil  xolumc  of  Ihc  sohilion   1  liter. 

'  (Juaiac  test,  pages  174,  lyi  and  .317. 

-  Test  for  free  acid,  jjage  120. 

■  Hammarsten's  reaction,  pages  152  and  },](). 

'  Hopkins-Cole  reat  tion,  page  8(;. 

'  Methods  for  determination  (jf  urea,  [lage  T,()i). 


APPENDIX.  415 

(b)  A  solution  of  sodium  hydroxide  having  a  specific  gravity  of 
1.25.    This  is  approximately  a  22.5  per  cent  solution. 

Preserve  both  solutions  in  rubber-stoppered  bottles  and  when 
needed  for  use  mix  one  volume  of  solution  a,  one  volume  of  solution  b, 
and  3  volumes  of  water. 

Iodine  Solution/ — Prepare  a  2  per  cent  solution  of  potassium 
iodide  and  add  sufhcient  iodine  to  color  it  a  deep  yellow. 

JoUes'  Reagent." — This  reagent  has  the  following  composition: 

Succinic  acid 40  grams. 

Mercuric  chloride 20  grams. 

Sodium  chloride 20  grams. 

Distilled  water    1000  grams. 

Kraut's  Reagent.^ — Dissolve  272  grams  of  potassium  iodide  in 
water  and  add  80  grams  of  bismuth  subnitrate  dissolved  in  200  grams 
of  nitric  acid  (sp.  gr.  1.18).  Permit  the  potassium  nitrate  to  crystallize 
out,  then  filter  it  off  and  make  the  filtrate  up  to  i  liter  with  water. 

LugoPs  Solution/ — Dissolve  4  grams  of  iodine  and  6  grams  of 
potassium  iodide  in  100  c.c.  of  distilled  water. 

Magnesia  Mixture/ — Dissolve  175  grams  of  magnesium  sulphate 
and  350  grams  of  ammonium  chloride  in  1400  c.c.  of  distilled  water. 
x^dd  700  grams  of  concentrated  ammonium  hydroxide,  mix  thoroughly, 
and  preserve  the  mixture  in  a  glass-stoppered  bottle. 

Millon's  Reagent/ — Digest  i  part  (by  weight)  of  mercury  with 
2  parts  (by  weight)  of  nitric  acid  (sp.  gr.  1.42)  and  dilute  the  resulting 
solution  with  2  volumes  of  water. 

Molisch's  Reagent/ — A  15  per  cent  alcoholic  solution  of 
a-naphthol. 

Molybdic  Solution/ — Molybdic  solution  is  prepared  as  follows, 
the  parts  being  by  iveight: 

Molybdic  acid i  part. 

Ammonium  hydroxide  (sp.  gr.  0.96)    4  parts. 

Nitric  acid  (sp.  gr.  i .  2)    15  parts. 

Moreigne's  Reagent/ — Combine  20  grams  of  sodium  tungstate, 
10  grams  of  phosphoric  acid   (sp.  gr.   1.13)   and   100  c.c.  of  water. 

'  Iodine  test,  page  45. 

-  Jolles'  reaction,  pages  96  and  310. 

^  Rosenheim's  bismuth  test  for  choline,  page  24S. 

■*  Gunning's  iodoform  test,  page  32?,  and  Bardach's  reaction,  page  92. 

"  Sodium  hydroxide  and  potassium  nitrate  fusion  method  for  determination  of  total 
phosphorus,  page  384. 

''  Millon's  reaction,  page  88. 

'  Molisch's  reaction,  page  22. 

*  Sodium  hydroxide  and  potassium  nitrate  fusion  method  for  determination  of  total 
phosphorus,  page  3S4. 

"  Moreigne's  reaction,  page  269. 


41 6  PHYSIOLOGICAL    CHEMISTRY. 

Boil  the  mixture  for  twenty  minutes,  add  water  to  make  the  volume 
of  the  solution  equivalent  to  the  original  volume,  and  acidify  with 
hydrochloric  acid. 

Morner's  Reagent/ — Thoroughly  mix  i  volume  of  formalin.  45 
volumes  of  distilled  water,  and  55  volumes  of  concentrated  sulphuric 
acid. 

Nakayama's  Reagent.- — Prepared  by  combining  99  c.c.  of  alcohol 
and  I  c.c.  of  fuming  hydrochloric  acid  containing  4  grams  of  ferric 
chloride  per  liter. 

Neutral  Olive  Oil.^ — Shake  ordinary  olive  oil  with  a  10  per  cent 
solution  of  sodium  carbonate,  extract  the  mixture  with  ether,  and 
remove  the  ether  by  evaporation.     The  residue  is  neutral  olive  oil. 

Nylander's  Reagent.* — Digest  2  grams  of  bismuth  subnitrate 
and  4  grams  of  Rochelle  salt  in  100  c.c.  of  a  10  per  cent  solution  of 
potassium  hydroxide.  The  reagent  should  then  be  cooled  and 
filtered. 

Obennayer's  Reagent.^ — Add  2-4  grams  of  ferric  chloride  to  a 
liter  of  hydrochloric  acid  (sp.  gr.  1.19). 

Oxalated  Plasma.*'— Allow  arterial  blood  to  run  into  an  equal 
volume  of  0.2  per  cent  ammonium  oxalate  solution. 

Para-dimethylaminobenzaldehyde  Solution.^ — This  solution  is 
made  by  dissolving  5  grams  of  para-dimcthylaminobenzaldchyde  in 
100  c.c.  of  10  per  cent  sulphuric  acid. 

Para-phenelenediamine  Hydrochloride  Solution.* — Two  grams 
dissolved  in  100  c.c.  of  water. 

Phenolphthalein." — Dissolve  i  gram  of  jjlunolphlhalein  in  100 
c.c.  of  95  per  cent  alcohol. 

Phenylhydrazine  Mixture.^" — This  mixture  is  prepared  by  com- 
bining I  part  of  phenylhydrazine-hydrochloride  and  2  parts  of  sodium 
acetate  by  uieighl.     These  are  thoroughly  mixed  in  a  mortar. 

Phenylhydrazine-acetate  Solution." — This  solution  is  prepared 
by  mixing  i  volume  of  glacial  acetic  acid,  i  \olume  of  water,  and  2 
volumes  of  phenylhydrazine   (the  base). 

'  Morner's  test,  page  82. 

^Nakayama's  reaction,  pagt-'s  151  and  jiy. 

•^  Emulsification  of  fats,  page  1.^3. 

*  Nylander's  test,  pages  2<)  and  306. 

*  Obermayer's  test,  page  27  s. 

*"'  Plxperiments  on  blood  plasma,  [)age  i')6. 

'  Ilertcr's  pjira-fJimclhylaminobenzaldehyde  reaction,  i)agc  r66. 

"  detection  of  hy<lrogen  peroxide,  page  221. 

'^  'I'opfer's  method,  page  40S. 

'"Phenylhydrazine  reaction,  pages  21,  and  30D. 

"  I'henylhydrazine  reaction,  pagi-s  21,  and  300. 


APPENDIX.  417 

Purdy's  Solution.^ — Purdy's  solution  has  the  following  composition : 

Cupric  sulphate    4-752  grams. 

Potassium  hydroxide   23  . 5  grams. 

Ammonia  (U.  S.  P.,  sp.  gr.  o . 9) 35o  •  o  c.c. 

Glycerol 38.0  c.c. 

Distilled  water,  to  make  total  volume  i  liter. 

Roberts'  Reagent.^ — -Mix  i  volume  of  concentrated  nitric  acid 
and  5  volumes  of  a  saturated  solution  of  magnesium  sulphate. 

Rosenheim's  lodo-Potassium  Iodide  Solution.^ — Dissolve  2 
grams  of  iodine  and  6  grams  of  potassium  iodide  in  100  c.c.  of  water. 

Salted  Plasma/ — Allow  arterial  blood  to  run  into  an  equal  vol- 
ume of  a  saturated  solution  of  sodium  sulphate  or  a  10  per  cent  solu- 
tion of  sodium  chloride.  Keep  the  mixture  in  the  cold  room  for  about 
24  hours. 

Schiff's  Reagent/ — This  reagent  consists  of  a  mixture  of  three 
volumes  of  concentrated  sulphuric  acid  and  one  volume  of  10  per 
cent  ferric  chloride. 

Schweitzer's  Reagent/ — Add  potassium  hydroxide  to  a  solution 
of  cupric  sulphate  which  contains  some  ammonium  chloride.  Filter 
off  the  precipitate  of  cupric  hydroxide,  wash  it,  and  bring  3  grams  of 
the  moist  cupric  hydroxide  into  solution  in  a  liter  of  20  per  cent  ammo- 
nium hydroxide. 

Seliwanoff's  Reagent/ — Dissolve  0.05  gram  of  resorcin  in  100 
c.c.  of  dilute  (1:2)  hydrochloric  acid 

Sherrington's  Solution/ — This  solution  possesses  the  following 
formula : 

Methylene-blue o .  i  gram. 

Sodium  chloride 1.2  grams. 

Neutral  potassium  oxalate 1.2  grams. 

Distilled  water    300 .  o  grams. 

Sodimn  Acetate  Solution/ — Dissolve  loo  grams  of  sodium 
acetate  in  800  c.c.  of  distilled  water,  add  100  c.c.  of  30  per  cent  acetic 
acid  to  the  solution,  and  make  the  volume  of  the  mixture  up  to  i  liter 
with  distilled  water. 

Sodium  Alizarin  Sulphonate/" — Dissolve  i  gram  of  sodium  aliz 
arin  sulphonate  in  100  c.c.  of  water. 

'  Purdy's  method,  page  364. 

^  Roberts'  ring  test,  pages  95  and  310. 

*  Rosenheim's  periodide  test,  page  24S. 

*  Experiments  on  blood  plasma,  page  196. 
^  Schiff's  reaction,  pages  155  and  247. 

^  Schweitzer's  solubility  test,  page  40. 
'  Seliwanoff's  reaction,  pages  34  and  333. 
^  "Blood  counting,"  page  20S. 
^  Uranium  acetate  method,  page  i^St,. 
^^  Topfer's  method,  page  40S. 
27 


41 8  PHYSIOLOGICAL    CHEMISTRY. 

Sodium  Sulphide  Solution.' — Saturate  a  i  per  cent  solution 
of  sodium  hydroxide  with  hydrogen  sulphide  gas  and  add  an  equal 
volume  of  i  per  cent  sodium  hydroxide. 

Solera's  Test  Paper.- — Saturate  a  good  quality  of  filter  paper 
with  0.5  per  cent  starch  paste  to  which  has  been  added  sufficient  iodic 
acid  to  make  a  i  per  cent  solution  of  iodic  acid  and  allow  the  paper  to 
dry  in  the  air.     Cut  it  in  strips  of  suitable  size  and  preserve  for  use. 

Spiegler's  Reagent,^ — This  reagent  has  the  following  composition: 

Tartaric  aciil 20  grams. 

Mercuric  chloride 40  grams. 

Glycerol 100  grams. 

Distilled  water    1000  grams. 

Standard  Ammonium  Thiocyanate  Solution.' This  sokition 
is  made  of  such  a  strength  that  i  c.c.  of  it  is  equal  to  i  c.c.  of  the  stand- 
ard argentic  nitrate  solution  mentioned  below.  To  prepare  the  solu- 
tion dissolve  12.9  grams  of  ammonium  thiocyanate,  NH^SCN,  in  a 
little  less  than  a  liter  of  water.  In  a  small  flask  place  20  c.c.  of  the 
standard  argentic  nitrate  solution,  5  c.c.  of  a  cold  saturated  solution  of 
ferric  alum  and  4  c.c.  of  nitric  acid  (sp.  gr.  1.2),  add  water  to  make 
the  total  volume  100  c.c,  and  thoroughly  mix  the  contents  of  the  flask. 
Xow  run  in  the  ammonium  thiocyanate  solution  from  a  burette  until 
a  permanent  brouni  tinge  is  produced.  This  is  the  end-reaction  and 
indicates  that  the  last  trace  of  argentic  nitrate  has  been  precipitated. 
Take  the  burette  reading  and  calculate  the  amount  of  water  necessary 
to  use  in  diluting  the  ammonium  thiocyanate  in  order  that  10  c.c.  of 
this  solution  may  be  exactly  equal  to  10  c.c.  of  the  argentic  nitrate 
solution.  Make  the  dilution  and  titrate  again  to  be  certain  that  the 
solution  is  of  the  proper  strength. 

Standard  Argentic  Nitrate  Solution.'^— Dissolve  29.06  grams  of 
argentic  nitrate  in  i  liter  (jf  distilled  water.  Each  cubic  centimeter  of 
this  solution  is  equivalent  to  0.0 r  gram  of  sodium  chloride  or  to  0.006 
gram  of  chlorine. 

Standard  Uranium  Acetate  Solution." — Dissolve  35.461  grams 
of  uranium  ucclate  in  1  liter  of  water.  One  c.c.  of  such  a  solution 
should  be  ef|ui\alent  to  0.005  Kram  of  P./).,,  phosjihoric  anhydride. 

'  Krliger  and  Si  hmidt's  mclhofi,  pages  _^6K  and  i,i)')- 

*  .S<jlera's  rea<  lion,  page  56. 

'  Spiegler's  ring  test,  pages  f;6  and  ^ro. 

*  Volhard-Arnold  method,  page  390,  and  Clark's  niodilK  alimi  of  Dclui's  niclhod, 
page  .^«X. 

'  Voiharri-Arnojd  method,  page  390,  Molir's  mctli(jd,  page  3S0,  ;i"d  Clark's  niodilu  alion 
of  Dehn's  method,  page  3SS. 

"  Uranium  a<  etale  method,  page  383. 


APPENDIX.  419 

This  solution  may  be  standardized  as  follows:  To  50  c.c.  of  a 
standard  solution  of  disodium  hydrogen  phosphate,  of  such  a  strength 
that  the  50  c.c.  contains  o.i  gram  of  P2O5,  add  5  c.c.  of  the  sodium 
acetate  solution  mentioned  on  p.  417  and  titrate  with  the  uranium 
solution  to  the  correct  end-reaction  as  indicated  in  the  method  proper 
on  p.  383.  Inasmuch  as  i  c.c.  of  the  uranium  solution  should  precipi- 
tate 0.005  gram  of  PjOj,  exactly  20  c.c.  of  the  uranium  solution  should 
be  required  to  precipitate  the  50  c.c.  of  the  standard  phosphate  solu- 
tion. It  the  two  solutions  do  not  bear  this  relation  to  each  other  they 
must  be  brought  into  the  proper  relation  by  diluting  the  uranium 
solution  with  distilled  water  or  by  increasing  its  strength. 

Starch  Iodide  Solution/ — Mix  o.i  gram  of  starch  powder  with 
cold  water  in  a  mortar  and  pour  the  suspended  starch  granules  into 
75-100  c.c.  of  boiling  water,  stirring  continuously.  Cool  the  starch 
paste,  add  20-25  grams  of  potassium  iodide  and  dilute  the  mixture 
to  50  c.c.  This  solution  deteriorates  upon  standing,  and  therefore 
must  be  freshly  prepared  as  needed. 

Starch  Paste." — Grind  2  grams  of  starch  powder  in  a  mortar 
with  a  small  amount  of  water.  Bring  200  c.c.  of  water  to  the  boiling- 
point  and  add  the  starch  mixture  from  the  mortar  with  continuous 
stirring.  Bring  again  to  the  boiling-point  and  allow  it  to  cool.  This 
makes  an  approximate  i  per  cent  starch  paste  which  is  a  very  satis- 
factory strength  for  general  use. 

Stokes'  Reagent.^ — A  solution  containing  2  per  cent  ferrous 
sulphate  and  3  per  cent  tartaric  acid.  When  needed  for  use  a  small 
amount  should  be  placed  in  a  test-tube  and  ammonium  hydroxide 
added  until  the  precipitate  which  forms  on  the  first  addition  of  the 
hydroxide  has  entirely  dissolved.  This  produces  ammonium  ferro- 
tartrate  which  is  a  reducing  agent. 

Suspension  of  Manganese  Dioxide/ — Made  by  heating  a  0.5 
per  cent  solution  of  potassium  permanganate  with  a  little  alcohol  until 
it  is  decolorized. 

Tanret's  Reagent/ — Dissolve  1.35  grams  of  mercuric  chloride 
in  25  c.c.  of  water,  add  to  this  solution  3.32  grams  of  potassium  iodide 
dissolved  in  25  c.c.  of  water,  then  make  the  total  solution  up  to  60  c.c. 
with  distilled  water  and  add  20  c.c.  of  glacial  acetic  acid  to  the 
mixture. 

'  Fehling's  method,  page  362. 

^  Fehling's  method,  page  362 

^  Hemoglobin,  page  loq.     Ha;mochromogen,  page  202. 

*  Krijger  and  Schmidt's  method,  pages  36S  and  399. 

^  Tanret's  test,  pages  g6  and  311. 


420  PHYSIOLOGICAL   CHEMISTRY. 

Tincture  of  Iodine.^ — Dissolve  70  grams  of  iodine  and  50  grams 
of  potassium  iodide  in  i  liter  of  95  per  cent  alcohol. 

Toison's  Solution.- — This  solution  has  the  following  formula: 

Methyl  violet    0-025  gram. 

Sodium  chloride i .  o  gram. 

Sodium  sulphate S .  o  grams. 

Glycerol 30.0  grams. 

Distilled  water    160.0  grams. 

Topfer's  Reagent.'' — Dissolve  0.5  gram  of  di-methylamino- 
azobenzene  in  100  c.c.  of  95  per  cent  alcohol. 

Tropaeolin  00.^ — Dissolve  0.05  gram  of  tropaeolin  OO  in  100  c.c. 
of  50  per  cent  alcohol. 

Uffelmann's  Reagent.^ — Add  a  5  per  cent  solution  of  ferric 
chloride  to  a  i  per  cent  solution  of  carbolic  acid  until  an  amethyst- 
blue  color  is  obtained. 

'  Smith's  test,  pages  152  and  319. 
^  "Blood  counting,"  page  208. 
^  Topfer's  method,  page  40S. 

*  Test  for  free  acid,  page  120. 

*  Uffelmann's  reaction,  page  125. 


INDEX. 


Acacia    solution,    formation    of    emulsion 

130 
Acetone,  299,  321 
formula  for,  321 

Gunning's  iodoform  test  for,  323 
Legal's  test  for,  323 
Lieben's  test  for,  323 
quantitative  determination  of,  393 
Reynolds-Gunning  test  for,  324 
Taylor's  test  for,  324 
Acholic  stool,  169 
Achroo-dextrins,  43,  54,  57 
CK-achroo-dextrin,  54 
^-achroo-dextrin,  54 
^'-achroo-dextrin,  54 
Acid,  acetic,  260,  285 

alloxyproteic,  259,  279,  336 
amino-acetic,  66 
amino-ethyl-sulphonic,  148,  236 
Ci:-amino-/J-hydroxy-propionic,  67 
a-amino-/?-imidazol-propionic,  72 
a-amino-iso-butyl-acetic,  74 
a-amino-methyl-ethyl-propionic,  7  s 
a-amino-normal  glutaric,  77 
a-amino-propionic,  67 
amino-succinic,  76 
a-amino-iso-valerianic,  73  (see  Valine) 

aspartic,  76 

benzoic,  157,  259,  284 

butjrric,  116,  220,  260,  285 

caproic,  213 

carbamic,  180 

cholic,  148 

chondroitin-sulphuric,  227,  259,  279 

citric,  213 

combined  hydrochloric,  58,  409 

cyanuric,  262 

a-£-di-amino-caproic,  76 

Qr-amino-/3-thiolactic,  disulphide  of,  70 

diaminotrihydroxydodecanoic,  62,  80 

diazo-benzene-sulphonic,  336 

ethereal  sulphuric,  158,  259,  273 

fatty,  128,  129,  134 

formic,  260,  285 

free  hydrochloric,  116,  120 

glutamic,  77 

glycocholic,  148 

glycuronic,  3S.  37 

glycerophosphoric,  244,  245,  260,  286 

glyoxylic,  89 

guanidine-a-amino-valerianic,  74 

hippuric,  IS7,  259,  276,  376 

homogentisic,  27,  239,  282 

indole-amino-propionic,  72 
indoxyl-sulphuric,  158,  274 


by,        Acid,  inosinic,  232,  237 
kynurenic,  259,  283 
lactic,  116,  125,  233 
lauric,  213 
mucic,  36,  40,  331 
myristic,  213 
nucleic,  104 
osmic,  247,  27s 
oxalic,  259,  279 
oxaluric,  259,  285 
oxymandelic,  259,  283 
oxyproteic,  259,  279,  336 
palmitic,  134 

para-cresol-sulphuric,  259,  273 
para-oxyphenyl-acetic,  158,  164,  259,  282 
para-oxyphenyl-a-amino-propionic,  68 
para-oxyphenyl-propionic,  158,     164,     259, 

282 
paralactic,  233,  260,  28s 
phenaceturic,  260,  286,  377 
phenol-sulphuric,  259,  274 
phenyl-a-amino-propionic,  68 
phosphocarnic,  232,  237,  260,  286 
phosphoric,  294 

pyrocatechin-sulphuric,  259,  274 
a-pyrrolidine-carboxylic,  78 
sarcolactic,  233 
skatole  acetic,  72 
skatole-carbonic,  164 
skatoxyl-sulphuric,  259,  274 
sulphaniUc,  336 
tannic,  45,  48 
taurocholic,  148 
uric,  27,  232,  259 
uroferric,  259,  279,  336 
uroleucic,  259, 

volatile  fatty,  158,  161,  260,  285 
Acid  albuminate.     See  Acid  metaprotein. 
Acid  infraprotein.     See  Acid  metaprotein. 
Acid  metaprotein,  108 

coagulation  of,  loS 
experiments  on,  108 
precipitation  of,  loS 
preparation  of,  108 
solubility  of,  108 
sulphur  content  of,  108 
Acidity    of    gastric    juice,    quantitative    deter- 
mination of,  40S 
urine,  cause  of,  252,  294 

quantitative  determination  of,  398 
Acidosis,  cause  of,  327 
Acid-hsmatin,  202 

Acree-Rosenheim  formaldehyde  reaction,  92 
Acrolein,  formation  of,  from  olive  oil,  132 
from  glycerol,  135 

421 


422 


INDEX. 


Activation,  5,  138 

Activation   by  calcium  salts,  139 

Adam's   paper   coil   method   for   determination 

of  fat  in  milk.  J04 
Adamkiewicz  reaction,  89 
Adenine,  237,  260 
Adipocere.  131 
Adler's  benzidine  reaction  for  bloodv  187.   192- 

317 
Agglutination,  191 
Alanine,  62,  67 
Albumin,  egg,  99 

powdered,  preparation  of,  99 

tests  on,  99 
serum,  84,  86.  178.  299.  308 
Albumin  in  urine,  299,  308 

acetic   acid    and    potassium   ferrocya- 

nide  test  for,  311 
coagulation  or  boiling  test  for,  310 
Heller's  ring  test  for,  308 
Jolles'  reaction  for,  310 
Roberts'  ring  test  for,  310 
sodium  chloride  and   acetic  acid   test 

for,  312 
Spiegler's  ring  test  for,  310 
Tanret's  test  for,  311 
tests  for.  308 
Albumins.  84,  86.  87 
Albuminates.     See  Metaproteins. 
Albuminates,  formation   of,    by    metallic    salts, 

93.  95 
Albuminoids,  84,  103 
Albumoscope,  95,  309 
Albumoses  (see  Proteoses,  p.  110) 
Alcohol-soluble  proteins.     See  Prolamins. 
Aldenyde,  20,  25 
Aldehyde  group,  38 
Aldehyde  test  for  alcohol,  42 
V.    Aider's    method    of    detecting    proteose    in 

unne,  314 
Aldose.  20 

Alkali  albuminate.     See  Alkali  metaprotein. 
Alkali-hamatin,  201 
Alkali  metaprotein,  108,  109 
experiments  on,  109 
precipitation  of,  108 
preparation  of.  108 
sulphur  content  of,  109 
Aliantoin.  259.  280 

crystalline  form  of,  280 
experiments  on,  281 
formula  for,  280 

preparation  of,  from  uric  acid,  281 
quantitative  rictermination  of,  401 
separation  of,  from  urine,  281 
Allen's  modification  of  Fehling's  test,  305 
Almt-n's  reagent,  preparation  of,  31s 
Alloxyproteic  acid,  259,  279,  336 
Aloin-turpentinc  test  for  "occult  blood,"   171. 

173 
Amandin,  84 
Amide  nitrogen,  61 
Amidulin.     See  Soluble  starch,  8,  S3 
Amino  acids,  62,  87,  158 

group,  86,  90 
rt-amjno-/y-hydroxy  propionic  acid,  67 
ar-amino-/'?-imida7,ol|)ropionic  acid,  77 


(i-amino-iso-butyl-acetic  acid,  74 
(V-aniino-normal-gKitaric  acid,  77 
Amino-succinic  acid.  76 
(V-amino-iso-valerianic  acid.  73 
Ammonia.  66,  103 
Ammonia  in  urine,  252,  260,  290 

quantitative   determination   of,   374 
Ammoniacal  silver  solution,  preparation  of,  401 
Ammoniacal-zinc     chloride     test    for    urobilin, 

288 
Ammonium    magnesium    phosphate     ("Triple 
phosphate"),  243,  295 
in  urinary  sediments,  339 
Ammonium  urate,  266,  342,  366 

crystalline  form  of,  Plate  VI,  opposite 
P-  343 
Amphopeptone,  1 1 1 
Amylase,  pancreatic.  139 

digestion  of  dry  starch  by,  139.  i4.S 

inulin  by,  145 
experiments  on,  8,  143 
influence  of  bile  upon  action  of,  144 

metallic  salts,  upon  action  of,  144 
most  favorable  temperature  for  action  of, 

144 
salivary,  53,  116 

activity  of,  in  stomach,  54,  116 
experiments  on,  8 
inhibition  of  activity  of,  54 
nature  of  action  of,  S4 
products  of  action  of,  54 
vegetable,  8 
Amylases,  3 

experiments  on,  8 
Amyloid,  48,  104 
Amylolytic  enzymes.      See  Amylases. 

quantitative     determination     of     ac- 
tivity of,  15 
Animal  jiarasites  in  feces,  170,  172 

in  urinary  sediments,  346,  355 
Anti-albumid,  1 19 
Anti-enzymes,  7 

experiments  on,  14 
Anti-pei)sin,  7,  14 
Antipcptone,  1 1 1 
Anti-rennin,  7 
Anti-trypsin,  7,  1  5 

Af)pendix,  411  * 

Arabinose,  20,  36 

orcin  test  on,  37 
phenylhydrazinc  test  on,  37 
Tollens'rcaclion  on,  37 
Arginine,  62,  74,  138 

Arnold-Lipliawsky    reaction    Im    diacctic    acid, 
326 
reagent,  preparation  of,  326 
Aromatic  oxyaeids,  259,  282 
Ascaris,  14,  IS 
Asparagine,  77 
Aspartic  acid,  62,  76,  138 

crystalline  form  of,  77 
formula  for,  76 
Ash    of    milk,    i|ii:iritil;aive    determination    of, 

407 
Assimilation  limit,  22 
Atkin.son  and  Kendall's  hicmin  test,  193 
Autolytic  enzymes,  3 


INDEX. 


423 


Bardach's  reaction,  92 

Barfoed's  reagent,  preparation  of,  12,  30,  307 
Barfoed's  test  for  monosaccharides,  30,  307 
Baryta  mixture,  preparation  of,  263 
Bayberry  tallow,  saponification  of,  133 

source  of,  133 
Bayberry  wax.     See  Bayberry  tallow,  133 
Beckmann-Heidenhain  apparatus,  256 
"  Bence  Jones'  protein,"  detection  of,  314 
Benedict's     method     for     quantitative     deter- 
mination of  sugar,  363 
Benedict's      method     for     quantative     deter- 
mination of  sulphur,  380 
Benedict's     method     for     quantitative     deter- 
mination of  urea,  373 
Benedict's  modifications  of  Fehling's  test,  27,  28 
solutions,  preparation  of,  27,  28 
solution,    for    use    in    quantitative    deter- 
mination of  sugar,  preparation  of,  363 
sulphur  reagent,  preparation  of,  380 
Benzidine  reaction,  Adler's,  for  blood,  187,  192  , 

317 
Benzoic  acid,  157,  259,  284 

crystalline  form  of,  284 
experiments  upon,  284 
formula  for,  283 
solubility  of,  284 
sublimation  of,  284 
Berthelot-Atwater  bomb  calorimeter,  382 
Bergell's   method    of   determination   of   ;9-oxy- 

butyric  acid,  397 
Bile,  147,  299,  318 

constituents  of,  148 
daily  secretion  of,  148 
freezing-point  of,  148 
influence  on  digestion,  gastric,  125 
pancreatic,  143,  144 
inorganic  constituents  of ,  148,  151 
nucleoprotein  of,  151 
reaction  of,  147,  151 
secretion  of,  147 
specific  gravity  of,  148 
Bile  acids,  148 

Guerin's  reaction  for,  153 
Hay's  test  for,  153  - 
Mylius's  test  for,  153 
Neukomm's  test  for,  153 
Pettenkofer's  test  for,  153 
tests  for,  153 

v.  Udransky's  test  for,  153 
Bile  acids  in  feces,  detection  of,  175 
Bile  acids  in  urine,  299,  320 

Hay's  test  for,  321 
Mylius's  test  for,  320 
Neukomm's  test  for,  321 
Pettenkofer's  test  for,  320 
tests  for,  320 

V.  Udransky's  test  for,  321 
Bile  pigments,  149 

Gmelin's  test  for,  151 
Hammarsten's  reaction  for,  152 
Huppert's  reaction  for,  152 
Rosenbach's  test  for,  151 
Smith's  test  for,  152 
test  for,  151 
Bile  pigments  in  urine,  299,  318 

Gmelin's  test  for,  318 


Bile  pigments  in  urine.  Hammarsten's  reaction 
for,  319 
Huppert's  reaction  for,  319 
Nakayama's  reaction  for,  319 
Rosenbach's  test  for,  318 
Salkowski's  test  for,  319 
Salkowski-Schipper's  reaction  for, 

320 
Smith's  test  for,  319 
tests  for,  318  J 

Bile  salts.  5.  148 

crystallization  of,   149,  154 
Biliary,  calculi,  151 

analysis  of,  154 
Bilicyanin,  149,  150 
Bilifuscin,  149 
Bilihumin,  149 
Biliprasin,  149 
Bilirubin,  149,  150 

crystalline  form  of,  150 
in  urinary  sediments,  339,  345  ii 

Biliverdin,  149,  150 
"Biological"  blood  test,  188 
Bismuth  test  for  choline,  248 
Biuret,  90,  262 

formation  of,  from  urea,  90,  264 
Biuret  potassium  cupric  hydroxide.      See  Cupri- 
potassium  biuret,  90 
test,  90 

Posner's  modification  of,  91 
Black's   method   for    determination    of   ^i3-oxy- 
butyric  acid,  396 
reaction  for  /?-oxybutyric  acid,  327 
reagent,  preparation  of,  327 
Blood,  178,  299,  315 

agglutination  of,  191 

Bordet  test  for,  188 

clinical  examination  of,  203 

coagulation  of,  187 

constituents  of,  178,  180 

defibrinated,  187 

detection  of,  187,  192,  198 

erythrocytes  of,  178,  180,  181 

experiments  on,  189 

form  elements  of,  178 

guaiac  test  for,  174,  188,  192 

hsemin  test  for,  187,  192 

oxyhemoglobin  of,  iSi 

"occult,"  in  feces,  171,  173 

in  urine,  299,  315 

leucocytes  of,  186 

medico-legal  tests  for,  187 

microscopical  examination  of,  189,  198 

nucleoprotein  of,  178.  179 

pigment  of,  181 

plaques,  178 

plasma,  178,  196 

preparation  of  hamatin  from,  195 

preparation  of  laky,  190 

quantitative  analysis  of,  410 

reaction  of,  178,  189 

serum,  179,  195 

specific  gravity  of,  178,  189 

spectroscopic  examination  of,  198 

test  for  iron  in,  190 

total  amount  of,  178 

V.  Zeynek  and  Nencki's  haemin  test  for,  193 


424 


INDEX. 


Blood  casts  in  urine,  351,  358 
Blood  corpuscles.  182,  184 

"counting,"  212 
Blood  dust,  1-8,  187 
Blood  in  urine,  299,  315 

Adler's  benzidine  reaction  for,  317 

guaiac  test  for,  317 

Teichmann's  haemin  test  for,  316 

Heller's  test  for,  316 

Heller-Teichmann  reaction  for,  316 

Schalfijew's  hamin  test  for,  3 1 7 

Schumm's  modification  of  guaiac  test 
for,  317 

spectroscopic  examination  of,  318 

tests  for,  316 

V.   Ze>-nek  and   Nencki's  Iwemin   test 
for,  317 
Blood  plasma,  178,  196 

constituents  of,  178 

crystallization   of   oxyhaemoglobin   of, 
181,  197 

effect  of  calcium  on  oxalated,  197 

experiments  on,  196 

preparation  of  fibrinogen  from,  197 
oxalated,  196 
salted,  197 
Blood  serum,   179,  19s 

coagulation  temperature  of,  19s 

constituents  of,  179 

experiments  on,  195 

precipitation  of  proteins  of,  195 

separation  of  albumin  and  globulin  of, 
196 

sodium  chloride  in,  196 

sugar  in,  196 
Blood  stains,  examination  of,  198 
Boas'  reagent,  as  indicator,  120 

preparation  of,  120 
Boekelman   and    Bouma's   method   for   deter- 
mination of  /5-oxybutyric  acid,  398 
Boettger's  test  for  sugar,  29 
Bomb  calorimeter,  Berthelot-Atwater,  382 
Bonanno's  reaction,  152,  320 
Bonanno's  reagent,  preparation  of,  152,  320 
Bone,  constituents  of,  229 

ossein  of,  preparation  of,  229 
Bone  ash,  scheme  for  analysis  of,  230 
Borchardt's  reaction  for  laevulose,  34,  333 
Bordet  test,  detection  of  human  blood  by,  188 
Boric  acid  and  borates  in  milk,  detection  of,  221 
Botlu's  reagent,  preparation  of,  24,  301 
Bottu's  test,  24,  301 
Buccal  glands,  52 
BufTy  coat,  formation  of,  180 
Bunge's  mass  action  theory,  1 16 
Butyric  acid,  116,  220 
Butyrin,  129 
Bynin,  84,  loj 

Cadaverin,  76 

Calcium  and  magnesium  in  urine,  260,  297 
carbonate  in  urinary  sediments,  339,  340 
casein,  i  iH 
oxalate,  339 

in  urinary  so'iimcnts,  339 
phosphate  in  urinary  sediments,  339 
in  milk,  219 


Calcium  sulphate  in  urinary  sediments,  339,  341 
Calculi,  biliary,  151,  154 

urinary,  357 

calcium  carbonate  in,  358 

oxalate  in,  358 
cholesterol  in,  360 
cystine  in,  358 
fibrin  in,  358 
indigo  in,  360 
phosphates  in,  358 
uric  acid  and  urates  in,  358 
urostealiths  in,  358 
xanthine  in,  358 
Calliphora,    larvae   of,    formation    of    fat    from 

protein  by,  132 
Cane  sugar  (see  Sucrose,  p.  40) 
Caproic  acid,  213 
Carbamic  acid,  180 
Carbohydrates,  20 

classification  of,  20 

composition  of,  20,  21 

review  of,  49 

scheme  for  detection  of,  50 

variation  in  solubility  of,  21 
Carbonates  in  urine,  260,  297 
Carbon  moiety  of  protein  molecule,  131 
Carbon  monoxide,  haemoglobin,  199 

tannin  test  for,  200 
Carboxyl  group.  86 
Camine,  232 
Carnitine,  232 

formula  for,  236 
Camomuscarine,  232 
Camosine,  232,  237 
Cartilage,  227 

constituents  of,  227 

experiments  on,  228 

Hopkins-Cole  reaction  on,  228 

loosely  combined  sulphur  in.  228 

Millon's  reaction  on.  228 

preparation  of  gelatin  from,  228 

solubility  of,  228 

xanthoproteic  test  on,  228 
Casein,  118,  214 

soluble,  118,  214 

calcium,  118,  214 

quantitative  determination  of,  407 
Caseinogen,  8s,  86,  105.  17s.  213.  218 

action  of  rennin  upon,  118,  214 

biuret  test  on.  219 

Millon's  test  on,  219 

precipitation  of.  218 

preparation  of.  218 

solubility  of.  219 

test  for  loosely  combined  sulphur  in.  219 

test  for  phosphorus  in.  219 
Casts,  346,  348 

blood.  346.  350 

epithelial.  346,  350 

fatty.  346,  351 

granular,  346,  350 

hyaline,  346,  349 

pus,  346.  3S3 

waxy,  346,  353 
Casts  in  urinary  sediments,  346,  348 
Cat  gut,  124 
Cataiase,  14 


INDEX. 


425 


Catalase,  experiments  on,  14 
Catalysis,  2 
Cellulose,  20,  48 

action  of  Schweitzer's  reagent  on,  49 
hydrolysis  of,  48 
iodine  test  on,  48 
solubiHty  of,  48 
Cellulose  group,  21 
Cerebrin,  244,  246 

experiments  on,  248 
hydrolysis  of,  248 
microscopical  examination  of,  248 
preparation  of,  248 
solubihty  of,  248 
Cerebro-spinal  fluid,  choline  in,  245 
Charcot-Leyden  cr>'stals,  171 

form  of,  170 
Chl<jrides  in  urine,  260,  293 
detection  of,  293 

quantitative   determination   of,    388 
Cholecyanin,  150 
Choleprasin,  149 

Cholera-red  reaction  for  indole,  165 
Cholesterol,  151,  155,  246 
crystalline  form  of,  155 
formula  for,  246 

iodine-sulphuric  acid   test  for,    155,   247 
isolation  of,  from  biliary  calculi,    154 
Liebermann-Burchard  test  for,  155,  247 
occurrence  of,  in  urinary  sediments,  339,  344 
preparation  of,  from  nervous  tissue,   247 
Salkowski's  test  for,  iss,  247 
Schifi's  reaction  for,  155,  247 
tests  for,  155,  247 
Choletelin,  149 
Choline,  245,  248 

formula    or,  245 
tests  for,  248 
Chondrigen,  104 
Chondroalbumoid,  227,  228 
Chondromucoid,  104,  227 
Chondroitin,  228 

Chondroitin-sulphuric  acid,  227,  259,  279 
Chondrosin,  228 

Chromoproteins,  see  Haemoglobins,  83,  86 
Cipollina's  test,  24,  301 
Clark's    modification    of    Dehn's    method    for 

determination  of  chlorides,  3 88 
Cleavage    products   of    protein    (see    Decompo- 
sition products),  60 
Clupeine,  85,  86 
Coagulated  proteins,  85,  109 
biuret  test  on,  no 
formation  of,  no 
Hopkins-Cole  reaction  on,  no 
Millon's  reaction  on,  no 
solubility  of,  no 
xanthoproteic  reaction  on,  no 
Coagulation  of  proteins,  98,  109 

changes  in  composition  during,   109 
fractional,  98,  109 
Coagulation   temperature   of   proteins,    98,    109 
apparatus  used  in  determining,   98 
method  employed  in  determining,   98 
Co-enzyme,  5 

Collagen,  84,  104,  223,  225 
experiments  on,  225 


Collagen,  percentage  of,  in  ligament,  227 
in  tendon,  224 
production  of  gelatin  from,   225 
solubihty  of,  225 
transformation  of,  225 
Colostrum,  216 

microscopical  appearance  of,  214 
Combined  hydrochloric  acid,  116 

tests  for,  120 
Compound  test  for  lactose  in  urine,  331 
Congeahng-point  of  fat,  136 
Congo  red,  as  indicator,  121 
preparation  of,  121 
Conjugated  proteins,  85,  86,  104 
classes  of,  85,  86,  104 
nomenclature  of,  85 
occurrence  of,  104 
Conjugate  glycuronates,  27,  299,  303,  328 

fermentation-reduction  test  for,  328 
Tollens'  reaction  on,  329 
Connective  tissue,  223 
Cowie's  guaiac  test,  174 
Creatine,  180,  232,  233,  259 
crystalline  form  of,  234 
formula  for,  236 

quantitative  determination  of,  387 
separation  of,  from  meat  extract,  240 
Creatinine,  27,  232,  259,  270 

coefficient,  definition  of,  270 
crystalHne  form  of,  271 
daily  excretion  of,  270 
experiments  on,  272 
formula  for,  236,  270 
Jalie's  reaction  for,  273 
quantitative  determination  of,  385 
Salkowski's  test  for,  273 
separation  of,  from  urine,  272 
Weyl's  test  for,  273 
Creatinine-zinc  chloride,  formation  of,  271,  272 
Cresol,  para,  158 
tests  for,  166 
Cross  and  Sevan's  reagent, 

preparation  of,  49 
'solubility  test,  49 
Cryoscopy,  255 
Cul-de-sac,  115 
Cupri-potassium  biuret,  formation  of,  90 

formula  for,  91 
Cyanuric  acid,  262 

formula  for,  262 
Cylindroids  in  urinary  sediments,  346,  353 
Cystine,  62,  70,  138 

crystalline  form  of,  71 
detection  of,  343 
formula  for,  70 

in  urinary  sediments,  339,  343 
Cytoglobulin  85,  86 
Cytosine,  105 

Wheeler- Johnson  reaction  for,  103 

Dakin's  methods  for  quantitative  determination 

of  hippuric  acid,  376 
Dare's  hasmoglobinometer,  205 
description  of,  205 

determination  of  haemoglobin  by,  205 
Darmstadter's    method    for    determination    of 
^-oxybutyric  acid,  397 


426 


INDEX. 


Deamidizing  enzyme,  3 
Decomposition  products  of  proteins,  60 
co'stalline  forms  of,  67-79 
experiments  on.  80 
isolation  of.  80 
Degradation  products  of  protein  (see  Decompo- 
sition products.  60) 
Dehn's  method,  Clark's  modification  of,  388 
Dehn's  reaction  for  hippuric  acid,  278 
Delusive  feeding  experiments.  115 
Derived  proteins,  85,  106 
Detection  of  preservatives  in  milk,  220 

boric  acid  and  borates.  221 

formaldehyde.  221 

hydrogen  peroxide,  221 

salicylic  acid  and  salicylates,  221 
Deuteroproteose,  86 
Dextrin.  21,47 

achroo  ,  43.  S3 

a-achroo  ,  53 

,3-achroo-,  53 

^-achroo-,  53 

er>-thro-,  43.  53 

action  of  tannic  acid  on.  48 

difTusibility  of.  48 

Fehling's  test  on.  48 

hydrolysis  of.  48 

iodine  test  on.  47 

solubility  of.  47 
Dextrosazone.   crystalline   form   of,   Plate    III, 

opposite  p.,  23 
Dextrose,  20.  22,  299 

Allen's  modification  of  Fehling's  test   for. 

30s 
Barfoed's  test  on,  30,  307 
Boettger's  test  on,  29,  306 
Bott  j's  test  on,  24.  301 
Cipollina's  test  on,  24,  301 
Benedict's  modification  of  Fehling's  test, 

27.  303 
difTusibility  of.  25 
experiments  on.  22 
Fehling's  test  on,  27.  303 
fermentation  of,  307 
iodine  test  on,  25 
Molisch's  reaction  on,  22 
Moore's  test  on,  25 
Nylander's  test  on,  29.  306 
phenylhydrazine  test  on,  23,  300 
riuantitative  determination  of,  362 
reduction  tests  on,  25,  302 
Ricgler's  reaction,  24,  301 
solubility  of.  22  . 
Trommcr's  test  on,  26,  302 
Dextrosazone,   crystalline   form   of,    Plate    III, 

opposite  p..  23 
Diacetic  acid.  299.  3^4 

Arnold-Lii>liawsky  test  for,  326 
formula  for,  324 
Gcrhardt's  test  for,  325 
quantitative  determination  of,  395 
Diamino  acid  nitrogen,  61 
Diaminotrihydroxydodecanoic  acid,  80 
a-£-diamino-caproic  acid,  76 
Diaxtase  (sec  Vegetable  amylase,  8) 
Diazo-bcnzcne-sulphonic  acid,  336 

reagent,  prejiaration  of,  336 


Diazo  reaction  (Ehrlich's).  336 

Differentiation  between  pepsin  and  pepsingen, 

123 

Digestion,  gastric,  115 
pancreatic,  137 
salivary,  52 

Di-methyl-amino-azobenzene    (see   Topfer's   re- 
agent), 120 

Dipeptides,  63,  65,  86 

Disaccharides,  38 

classification  of,  20 

Dissociation   products  of   protein    (see   Decom- 
position products,  66) 

Doremus-Hinds  ureometer,  372 

Drying  method  for  determination  of  total  solids 
in  urine,  402 

Duodenum,  epithelial  cells  of.  137 

Earthy  phosphates  in  urine,  294.  296 

quantitative   determination   of,    384 
Edestan,  17.  8s,  106 

experiments  on,  106 
Edestin,  84,  100 

coagulation  of,  102 

crystalline  forffs  of,  101 

microscopical  examination  of,  102 

Millon's  test  on,  102 

preparation  of,  101 

solubility  of,  102 

tests  on  crystallized,  102 
filtrate  of,  102 
Ehrlich's  diazo-benzene-sulphonic  acid  reagent, 

preparation  of,  336 
Ehrlich's  diazo  reaction,  336 
Ehrlich's  mechanical  eye-piece,  use  of,  211 
Einhorn's  saccharometcr,  3 1 
Elastin,  84,  104,  226 

exi)eriments  on,  227 

I)reparation  of,  227 

solubility  of,  227 
Electrical  conductivity  of  urine,  257 
Embryos,  glycogen  in,  233 
Enterokinase,  s,  138 
Enzymes,  i 

activation  of,  s 

absorption  of,  4 

classification  of,  3 

definition  of,  i 

experiments  on,  8 

preparation  of,  4 

pro7)crties  of,  4 
lipiguanine,  260,  289 
li|)isarkine,  260,  289 
Epithelial  cells  in  urinary  sediments,  346 

casts  in  urinary  sediments,  346,  350 
Epithelial  tissue,  223 

experiments  on,  223 
Erepsin.  140 

experiments  on,  13 
Erythrocytes.  178,  181 

counting  the,  208 

diameter  of,  180 

form  of,  180  / 

influence  of  osmotic  pressure  on,  i')i 

in  urinary  sediments,  346,  354 

number  of,  jier  cubic  mm.,  181 

of  different  species,  180 


INDEX. 


427 


Erythrocytes,  stroma  of,  181 

variation  in  number  of,  181 
Erythro-dextrin,  43,  53 
Esbach's  albuminometer,  362 

method  for  determination  of  albumin,  361 

reagent,  preparation  of,  361 
Ester,  definition  of,  128 

hydrochloric  acid,  of  ha;matin,  195 

sulphuric  acid,  of  hasmatin,  195 
Ethereal  sulphates,  273,  291 

quantitative  determination  of,  379 
Ethereal  sulphuric  acid,  158,  259,273 
Ethyl  butyrate  test  for  pancreatic  lipase,  145 
Euglobulin,  179 
Excelsin,  loi 

crystalline  form  of,  103 
Extractives  of  muscular  tissue,  232 
nitrogenous,  232 
non-nitrogenous,  232 

Fatigue  substances  of  muscle,  236 
Fats,  128 

absorption  of,  130 

apparatus    for    determination    of   melting- 
point  of,  13s 
chemical  composition  of.  128,  130 
congealing-point  of,  136 
crystallization  of,  130,  133 
digestion  of,  130 
emulsification  of,  133 
experiments  on,  132 
formation  of  from  protein,  131 
formation  of  acrolein  from,  132 
hydrolysis  of,  129 
in  milk,  213,  220 
in  urine,  299,  330,  404 
melting-point  of,  136 
nomenclature  of,  129 
occurrence  of,  128 
permanent  emulsions  of,  133 
quantitative  determination  of,  in  milk,  404 
rancid,  130 
reaction  of,  130 
saponification  of,  129,  133 
solubility  of,  130,  132 
transitory  emulsions  of,  133 
Fat-splitting  enzymes  (see  Lipases  3,  9) 
Fatty  acid,  128,  129,  134 
Fatty  casts  in  urinary  sediments,  346,  351 
Fatty  degeneration,  131 
Feces,  168 

blood  in,  171 

daily  excretion  of,  168 

detection  of  albumin  and  globulin  in,  176 

bile  acids  in,  175 

bilirubin  in,  175 

caseinogen  in,  175 

cholesterol  in,  173 

hydrobilirubin  in,  174 

inorganic  consituents  of,  176 

nucleoprotein  in,  176 

proteose  and  peptone  in,  176 
experiments  on,  172 
form  and  consistency  of,  170 
macroscopic  constituents  of,  170 
microscopic  constituents  of,  170 
odor  of,  169 


Feces,  pigment  of,  168 
reaction  of,  169 

separation  of,  importance  of,  170 
Fecal  bacteria,  171 

Fehling's  method  for  determination  of  dextrose, 
362 
Benedict's    modification    of, 
363 
solution,  preparation  of,  27,  303 
test,  27,  303 

Allen's  modification  of,  305 
Benedict's  modifications  of,  27,  303 
Ferments,  classification  of,  i 
Fermentation  tost,  30,  307 
Fermentation     method     for     determination     of 

dextrose,  365 
Fermentation-reduction      test      for      conjugate 

glycuronates,  328 
Ferric  chloride  test  for  thiocyanate  in  saliva,  56 
Fibrin,  179,  187,  i97<  299 

in  urinary  sediments,  346,  356 
separation  of,  from  blood,  187,  197 
solubility  of,  197 
Fibrin  ferment,  180,  187 
Fibrinogen,  179,  187 
Fibroin,  silk,  65 
Fischer  apparatus,  70 

photograph  of,  70 
Fleischl's  haemometer,  203 
description  of,  203 

determination  of  haemoglobin  by,  203 
Fleischl-Miescher  hsmometer,  204 
Fluorides  in  urine,  260,  298 
Fly-maggots,  experiments  on,  131 
Folin-Hart  method  for  determination  of  com- 
bined acetone  and  diacetic  acid,  391 
for  determination  of  diacetic  acid.  395 
Folin-Messinger-Huppert     method     for     deter- 
mination of  diacetic  acid,  395 
Fohlin's   method  for  determination  of  acetone, 
393 
acidity  of  urine,  398 
ammonia,  374 
creatinine,  385 
ethereal  sulphates,  379 
inorganic  sulphates,  379 
total  sulphates,  378 
urea,  371 
Folin-Benedict   and  Myers'    method   for   deter- 
mination of  creatine,  387 
Folin-Shaffer  method  for  determination  of  uric 

acid,  366 
Foreign  substances  in  urinary  sediment,  346,  356 
Formation  of  methylphenyllsevulosazone,  35 
Form  elements  of  blood,  178 
Formic  acid,  260,  285 
Fractional  coagulation  of  proteins,  109 
Free  hydrochloric  acid,  116 
tests  for,  120 
Freezing-point  of  bile,  14S 
blood,  178 
milk,  213 

pancreatic  juice,  138 
urine,  255 
Fuchsin-frog  experiment,  238 
Fuld  and  Levison's  method  for  peptic  activity  , 
17 


428 


INDEX. 


Fundus  glands.  115 

Furfurol  solution,  preparation  of,  153 

Fusion  mixture,  preparation  of,  100 

Galactase,  316 
Galactose,  20,  35,  399,  332 

experiments  on,  35 
Gallic  acid  test  for  formaldehyde,  220 
Gan?LSsini's  test.  269 
Gastric  digestion,  115 

conditions  essential  for,  122 

general  experiments  on,  122 

influence  of  bile  on,  125 

influence  of  different  temperatures  on, 

123 
most  favorable  acidity  for,  123 
power  of  different  acids  in,  124 
products  of,  119 
Gastric  fistula,  115 
Gastric  juice,  115 

acidity  of,  116 

artificial,  preparation  of,  119 
composition  of,  116 
enzymes  of,  116 

origin  of  hydrochloric  acid  of,  116 
quantitative  analysis  of,  408 
quantity  of,  115 
reaction  of,  116 
specific  gravity  of,  116 
lactic  acid  in,  test  for,  125 
Gastric  lipase,  116,  118 
Gastric  protease,  i 
Gastric  rennin,  116,  118,  125 

action  of,  upon  caseinogen,   118,  214, 

218 
experments  on,    125,  218 
influence  of,  upon  milk,  125,  218 
in  gastric  juice,  absence  of,  118 
nature  of  action  of,  118 
occurrence  of,  118 
Gelatin,  84,  224,  226 
coagulation  of,  226 
experiments  on,  226 
formation  of,  225 
Hopkins-Cole  reaction  on,  226 
Millon's  reaction  on,  226 
precipitation  of,  by  alcohol,  226 
alkaloidal  reagents,  226 
metallic  salts,  326 
precipitation  of,  by  mineral  acids,  226 
preparation  of,  from  cartilage,  228 

from  collagen,  225 
Balting-out  of,  226 
solubility  of,  226 
Gerhardt's  test  for  diacetic  acid,  325 
Gerhardt's  test  for  urobilin,  288 
Gics'  biuret  reagent,  preparation  of,  91 
Gliadin,  84,  103 
Globin,  84 

Globulins,  84,  94,  100 
experiments  on,  loi 
preparation  of,  10 1 
serum,  84,  178,  299,  312 
in  urine,  399,  31  2 
tests  for,  3 1 2 
vegetable,  84 
Glucoproteins  (see  Glycoproteins,  p.  8s) 


Glucose  (see  Dextrose,  p.  22) 
Glutamic  acid,  62,  77,  138 

formula  for,  77 
Glutelins,  84,  102 
Glutenin,  84,  102 
Glycerol,  129,  135 

borax  fusion  test  on,  135 

experiments  on,  135 

formula  for,  129 
Glycerol  extract  of  pig's  stomach,  preparation 

of,  119 
Glycerophosphoric  acid,  224,  243,  260,  286 
Glycocholic  acid,  148 
Glycocholic  acid  group,  148 
Glycocoll,  61,  6s,  148 

formula  for,  65,  148 

preparation  of,  156 
Glycocoll  ester  hydrochloride,  crystalline  form 

of,  66 
Glycogen,  20,  4S.  232.  233 

experiments  on,  240 

hydrolysis  of,  240 

in  embryos,  233 

influence  of  saliva  on,  240 

iodine  test  on,  240 

preparation  of,  240 
Glycoproteins,  85,  104,  224 

experiments  on,  225 

hydrolysis  of,  225 
Glycosuria,  alimentary,  21 
Glycuronates,  conjugate,  26,  300,  304,  329 
Glycuronic  acid,  35 
Glycyl-glycine.  formation  of,  64 
Glyoxylic  acid,  89 

formula  for,  89 
Gmelin's  test  for  bile  pigments,  151,  319 

Rosenbacb's  modification  of,  152,  319 
Granular  casts  in  urinary  sediments,  346,  351 
Granulose,  41 
Green  stools,  cause  of,  169 
Gross'  method  for  quantitative   determination 

of  tryptic  activity,  19 
Guaiac  solution,  preparation  of,  412 
Guaiac  test  on  blood,  174,  188,  192 
on  feces,  174 

milk,  218 
in  urine,  317 
Guaiac  test,  Schumm's  modification  of,  192 
Guaiac  test  on  pus,  349 
Guanidinc-a-amino-valerianic  acid,  73 
Guanidine-residue,  60 
Guanine,  232,  237 

Gums  and  vegetable  mucilage  group  of  carbo- 
hydrates, 20 
Gunning's  iodoform  test  for  acetone,  325 
Gunzberg's  reagent,  as  indicator,  120 

preparation  of,  120 
Gurber's  reaction  for  indican,  276 

H.'ematin,  106 

acid-,  202 

alkali-,  203 

preparation  of,  195 

reduced  alkali-,  302 
Hx-matodin,  150,  170 

crystalline  form  of,  150,  170 

in  urinary  sediments,  339,  345 


INDEX. 


429 


Hsematuria,  316 

Ifematoporphyrin,  10,  186,  203,  300,  307 

in  urine,  300,  331 
Hsemin  crystals,  form  of,  194 

test,  193 
Hsmochromogen,  106,  iSi,  202 
Hsemocyanin,  85,  87,  106 
Hsemoconein  (see  Blood  dust,  178,  187) 
Haemoglobin,  85,  87,  104,  105 
carbon  monoxide,  186,  201 
decomposition  of,  181 
diffusion  of,  192 
met,  186,  202 
oxy,  181,  186,  199 
quantitative  determination  of,  203 
reduced,  199 
Haemoglobins,  85,  105 
Hemoglobinuria,  316 
Hammerschlag's     method     for     determination 

of  specific  gravity  of  blood,  190 
Hammarsten's  reaction,  152,  320 

reagent,  preparation  of,  152,  320 
Hay's  test  for  bile  acids,  155,  321 
Heintz  method  for  determination  of  uric  acid, 

168 
Helicoprotein,  85 

HeUer's  test  for  blood  in  urine,  316 
HeUer-Teichmann  reaction  for  blood  in 'urine, 

317 
HeUer's  ring  test  for  protein,  95,  309 
Hemi-cellulose,  20 
Herter's  naphthaquinone    reaction    for    indole, 

164 
Herter's    para-dimethylaminobenzaldehyde    re- 
action, 166 
Heteroproteose,  87 
Heteroxan thine,  260,  289 
Hexone  bases,  75 
Hexoses,  19,  20 

Hippuric  acid,  156,  157,  259,  276  377 
cr\'staUine  form  of,  277 

Dakin's    method    for    quantitative    deter- 
mination of,  377 
Dehn's  reaction  for,  278 
experiments  on,  156,  277 
formula  for,  157 
in  urinary  sediments,  344 
melting-point  of,  278 
Roaf's  method  for  crystallization  of, 

278 
separation  of,  from  urine,  277 
solubility  of,  278 
Hippuric  acid,  sublimation  of,  279 

synthesis  of,  156 
Histidine,  61,  72,  138 

hydrochloride,  crji-stalline  form  of,  72 
Knoop's  color  reaction  for,  72 
Histones,  84,  86 

Hoffmann's  reaction  for  tyrosine.  Si 
Homogentisic  acid,  26,  283 

formula  for,  283 
Hopkins-Cole  reaction,  89 

on  solutions,  89 
on  solids,  99 
Hopkins-Cole  reagent,  preparation. of,  89 
Hopkins-Cole  reagent  (Benedict  modification), 
preparation  of,  90 


Hordein,  77,  84,  103 

Horismascope  (see  Albumoscope,  95) 

Hormone,  definition  of,  137 

Hopkins'  thiophene  reaction  for  lactic  acid,  126 

Hiifner's  urea  apparatus,  372 

Human  fat,  composition  of,  130 

Huppert's  reaction  for  bile  pigments,  152,  319 

Hurthle's  experiment,  243 

Hyaline  casts  in  urinary  sediments,  346,  350 

Hydrobilirubin,  detection  of,  in  feces,  175 

extraction  of,  175 
Hydrochloric  acid  of  the  gastric  juice,  116 

origin  of,  theories  as  to,  116 
Hydrochloric     acid     test     for     formaldehyde 

(Leach),  221 
Hydrogen  peroxide  in  urine,  260,  299 

detection  of,  in  milk,  222 
Hydrolysis  of  cellulose,  47 

cerebrin,  248 

dextrin,  46 

glycogen,  240 

inulin,  45 

proteins,  62 

starch,  44 

sucrose,  39 
Hyperacidity,  116 
Hypoacidity,  116 

Hypobromite  solution,  preparation  of ,  370 
Hypoxanthine,  232,  241.  266,  289 

formula  for,  237 
Hypoxanthine   silver  nitrate,    cr\-stalline   form 
of,  241 

IchthuHn,  85 
Ignotine,  232 

formula  for,  237 
Imide  bonds,  64 
Indican,  158,  274,  307 

formula  for,  159,  275 

Giirber's  reaction  for,  276 

Jafle's  test  for,  275 

LaveUe's  reaction  for,  276 

Obermayer's  test  for,  276 

origin  of,  158,  274 

Rossi's  reaction  for,  276 
Indigo-blue,  159,  27s 

formula  for,  159,  275 
Indigo  in  tuinary  sediments,  339,  346 
Indole,  158 

formula  for,  158 

origin  of,  158 

test  for,  164 
Indole-amino-propionic  acid,  71 
Indoxyl,  158,  274,  275 

formula  for,  158,  275 
Indoxyl,  origin  of,  159,  274 

potassium  sulphate  (see  Indican,  pp.   158, 
159,  274  ) 
Indoxyl-stilphuiic  acid,  158,  275 

formula  for,  15 8,  275 
Infraproteins  (see  Metaproteins,  85) 
Inorganic  physiological  constituents  of  urine,  290 
Inosinic  acid,  232 

formula  for,  237 
Inosite,  20,  300,  334 

formula  for,  334 
in  urine,  300,  334 


43° 


INDEX. 


Intestinal  juice.  140 

enzymes  of,  140 
preparation  of.  140 
Inulase,  45 
Inulin,  21.  44 

action  of  amylolytic  enzymes  on,  45,  56 

Fehling's  test  on,  45 

hydrolysis  of,  45 

iodine  test  on,  45 

reducing  power  of.  44 

solubility  of.  44,  45 

sources  of.  44 
Inversion,  39,  41 

Invertases.  experiments  on,  11,  141 
Invertin  (see  Sucrase,  p.  39) 
Inverting  enzymes,  3 
Invert  sugar.  39.  141 
Iodide  of  dextrin.  46 

of  starch.  42 
Iodine  test,  24,  42,  45,  46 
Iodine-sulphuric  acid  test  for  cholesterol,   154, 

247 
lodoform  test  for  alcohol,  40 
lodothymol  compound.  324 
Iron  in  blood,  185,  191 

detection  of,  191 

in  bone  ash,  230 

detection  of,  230 
Iron  in  protein.  60 
Iron  in  urine,  261,  298 

detection  of,  299 
Isoleucine,  75 
Isomaltose,  20.  38,  53 

Jaffe's  reaction  for  creatinine,  273 

Jaflfe's  test  for  indican,  275 

V.  Jaksch-Pollak  reaction  for  melanin,  336 

Jejunum,  epithelial  cells  of,  137 

Jolles'  reaction  for  protein,  96,  311 

reagent,  preparation  of.  96.  311 
Juice,  gastric.  1:5-119 

pancreatic.  137-140 

intestinal.  140 

Kastle's  peroxidase  reaction.  218 
Kephalin,  244,  246 
Kephyr,  38 
Keratin,  84,  223 

e:;periments  on,  223 
soluVjility  of,  223 
sources  of,  223 
sulphur  content  of,  223 
Ketone.  20,  25 
Kctose,  20 
Kjcldahl  methoil  for  determination  of  nitrogen, 

375 
Knoop's  coloi  reaction  for  histidine,  72 
Knop-Hufner  hypobromite  methorl   for  (k-tcr- 

mination  of  urea,  369,  371 
Konto's  reaction  for  indole.  165,  177 
KAppe's  electrolytic  dissociation  theory,  116 
Koumyss,  38 

Kraut's  reagent,  preparation  of,  249 
Kniger  and  Schmidt's  method  for  the  quanti- 
tative    determination 
of  purine  bases,  400 
of  uric  acid,  368 


Kiilz's  test  for  ^-oxybutyric  acid,  338 
Kwilecki's  modification  of  Esbach's  method,  362 
Kynurenic  acid,  259,  283 
formula  for,  283 
isolation  of,  from  urine,  284 
quantitative  determination  of,  284 

Lactalbumin,  84,  214,  217 

quantitative  determination  of,  406 
Lactase,  12,  138,  140 

experiments  on,  12 
Lactic  acid,  38,  126,  232 

ferric  chloride  test  for,  126 
Hopkins'  thiophene  reaction  for,  126 
in  muscular  tissue,  232,  233 
in  stomach  contents,  126,  127 
tests  for,  127 
Utfelmann's  test  for,  127 
Lacto-globulin,  214,  217 
Lactometer,   determination   of   specific  gravity 

of  milk  by,  404 
Lactosazone,    crystalline    form    of,    Plate    III, 

opposite  p.  23 
Lactoscope,  Feser's,  405 
Lactose.  20,  38 

experiments  on.  39 

fermentation  of,  38 

in  urine,  300,  331 

quantitative  determination  of,  406 
Lactosin  in  milk,  217 
Laevo-a-proline,  78 
Laevulosazone,    crystalline   form    of,    Plate    III, 

opposite  p.  23 
Leevulose,  20,  33 

Borchardt's  reaction  for.  33 

in  urine,  300,  333  * 

methyl-phenylhydrazine  test  for,  34 

Seliwanoff's  reaction  for,  34 
Laiose  in  urine,  300,  335 
Laked  blood,  178,  189 
Laky  blood,  191 
Laurie  acid,  214 
Laurin,  129 

Lavelle's  reaction  for  indican,  276 
Leach's  hyilrochloric  acid  test  for  formaldehyde, 

221 
Lecithans,  85 
Lecithin,  148,  244,  246 

acrolein  test  on,  247 

decomposition  of,  245 

experiments  on,  24s 

formula  for,  245 

microscopical  examination  of,  246 

oiimic  acid  test  on,  246 

preparation  of,  246 

test  for  phosphorus  in,  247 
Lccithoproteins,  85,  106 
Legal 's  reaction  for  indole,  165 

test  for  acetone,  324 
Leucine,  61,  73  74,  138,  180 

crystalline  form  of  impure,  34s 
pure,  74 

experiments  on,  82 

formula  for,  74 

in  urinary  sediments,  339,  344 

microscopical  examination  of,  82 

separation  of,  frr)m  tyrosine,  80 


INDEX. 


431 


Leucine,  solubility  of,  82 

sublimation  of,  82 
Leucocytes,  178,  186 

counting  the,  211 

number  of,  per  cubic  mm.,  186 

size  of,  186 

variation  in  number  of,  186 
Leucocytosis,  186 
Leucosin,  94 

Leucyl-alanyl-glycine,  formation  of,  64 
Leucyl-leucine,  formation  of,  64 
Lichenin,   21,   46 
Lieben's  test  for  acetone,  324 
Lieberkiilin's  jelly   (see  Alkali  metaprotein,   p. 

108) 
Liebermann-Burchard      test     for     cholesterol, 

IS4.  245 
Liebermann's  reaction,  91 
Lipase,  gastric,  118 
Lipase,  pencreatic,  10,  129 
experiments  on,  10 
ethyl-butyrate  test  for,  146 
litmus-milk  test  for,  145 
Lipases,  3,  10 

experiments  on,  10 
Lipoids  of  nervous  tissue,  243,  245 
Lipolytic  enzymes  (see  Lipases,  p.  145) 
"Litmus-milk"  test  for  pancreatic  lipase,  145 
Lugol's  solution,  preparation  of,  92 
Lysine,  61,  75,  138 
Lysine  picrate  crystall'iie  form  of,  76 

Magnesia  mixture,  preparation  of,  290 
Magnesium  in  urine,  260,  298 

phosphate  in  uiinary  sediments,  339,  34s 
Maltase,  13,  37,  53,  141 

experiments  on,  13 
Maltosazone,    crystalline    form    of,    Plate    III, 

opposite  p.  23 
Maltose,  21,  37 

experiments  on,  38 

structure  of,  37 
Marshall's  urea  apparatus,  370 
Melanin  in  urine,  300,  335 

urinary  sediments,  339,  346 
Melting-point  apparatus,  135 

of  fats,  determination  of,  136 
Messinger-Huppert,   method  for  determination 

of  combined  acetone  and  diacetic  acid,  393 
Metaproteins,  85,  106,  107 

acid,  85 

alkali,  85 

experiments  on,  108 

precipitation  of,  108 

sulphur  content  of,  108 
Methasmoglobin,   186,   202 
Methylene  blue,  124 
Methyl-mercaptan,  158,  169 
Methyl-pentose  (see  Rhamnose,  p.  21) 
Methylphenylhydrazine,  34 
Methylphenyllaevulosazone,  formation  of,  33 
i-methylxanthin,  260,  289 
Mett's    method    for    determination    of    peptic 

activity,  17 
Mett's  tubes,  preparation  of,  18 
Micro-organisms  in  urinary  sediments,  346,  356 
Milk,  214 


Milk,  citric  acid  in,  214 

detection  of  calcium  phosphate  in,  220 
lactose  in,  221 
preservatives  in,   221 

difference  between  human  and  cow's,  215 

experiments  on,  217 

formation  of  film  on,  214.  217 

feezing-point  of,  214 

guaiac  test  on,  218 

influence  of  rennin  on,  125 

isolation  of  fat  from,  221 

Kastle's  peroxidase  reaction  of,  218 

microscopical  appearance  of,  215,  217 

preparation  of  caseinogen  from,  219 

properties  of  caseinogen  of,  219 

quantitative  analysis  of,  404 

reaction  of,  214,  217 

separation  of  coagulable  proteins  of,  220 

specific  gravity  of,  214,  217 
Millon's  reaction,  88 

reagent,  preparation  of,  89 
Mohr's  method  for  determination  of  chlorides, 

389 
Molisch's  reaction,  22 
Molybdic  solution,  preparation  of,  55 
Monamino  acid  nitrogen,  60 
Monosaccharides,  20,  21 

Barfoed's  test  for,  30,  308 

classification  of,  20 
Moreigne's  reaction  for  uric  acid,  270 

reagent,  preparation  of,  270 
Morner-Sjoqvist-Folin    method    for    determina- 
tion of  urea,  373 
Morner's  reagent,  preparation  of,  82 

test  for  tyrosine,  82 
Motor  and  functional  activities  of  the  stomach , 

I2S 

Mucic  acid,  34,  38,  332 

test,  34,  39,  332 
Mucin,  54,  8s,  87 

biuret  test  on,   54 
hydrolysis  of,  55 
isolation  of,  from  saliva,  54 
Millon's  reaction  on,  54 
Mucins,  85,  87 
Mucoid,  85,  104,  224 

experiments  on,  225 
hydrolysis  of,  225 
in  urine,  285,  315 
preparation  of,  from  tendon,  224 
Mucoids,  85,  87 
Murexide  test,  269 
Muscle  plasma,  231,  238 

formation  of  myosin  clot  in,  231 
fractional  coagulation  of,  231,  23S 
preparation  of,  237.  238 
reaction  of,  233,  238 
Muscular  tissue,  231 

commercial  extracts  of,  236 
experiments  on  "dead,"  239 

"living,"  237 
extractives  of,  232,  237 
fatigue  substances  of,  236 
formulas    of    nitrogenous    extractives 

of.  237 
glycogen  in,  232,  240 
involuntary,  231 


432 


INDEX. 


Muscular  tissue,  lactic  acid  in,  234,  236,  239 
nonstriated,  231 
pigment  of,  336 
preparation  of  glycogen  from,  240 

muscle  plasma  from,  238 
proteins  of,  231 
reaction  of  living,  234 
separation  of  extractives  from,  241 
striated,  231 
voluntary-.  231 
Myohiemain,  236 
Myosan,  85 

formation  of,  239 
Myosin,  231 

biuret  test  on,  239 
coagulation  of,  239 
preparation  of,  239 
solubility  of,  239 
Myosinogen,  231 
Myristic  acid,  214 
Myristin,  130 
Myrtle  wax  (see  Bayberry  tallow,  133) 

Nakayama's  reaction  for  bile  pigments,  152,  319 

reagent,  preparation  of,  152,  319 
Nencki  and  Sieber's  reaction  for  urorosein,  336 
Neosine,  232 

formula  for,  237 
Nervous  tissue,  244 

constituents  of   244 
experiments  on  lipoids  of,  246 
lipoids  of,  244,  246 
percentage  of  water  in,  244 
phosphorized  fats  of,  244 
proteins  of,  244 
Neurokeratin,  244 

Neutral  olive  oil,  preparation  of,  133 
Neutral  sulphur  compounds,  259,  280 
Nitrates  in  urine,  260,  299 
Nitrites  in  saliva,  test  for,  55 
Nitrogen,  60 

forms  of  in  protein  molecule,  60 
importance  of,  in  sustaining  life,  60 
in  urine,  quantitative  determination  of,  37s 
Nitrogen  iodide,  formation  of,  323 
Nitrogenous  extractives  of  muscular  tissue,  232 

formulas  for,  236 
Nitroso-indole  nitrate  test,  165 
Nitrosothymol,  formation  of  in  Heller's  test,  310 
Non-nitrogenous  extractives  of  muscular  tissue, 

231 
Normal  urine,  249 

characteristics  of,  249 
constituents  of,  259 
experiments  on,  259,  299 
Novaine,  232 

formation  for,  237 
Nubecula,  285,  31S 
Nucleic  acid,  85,  105 
Nucleins,  105,  iiy,  244 
Nuclfohistonc,  85,  87 
NiKlfriprotcins,  85,  87,  104,  244,  260 
in  bile,  i  s  i 
in  feces,  176 
in  nervous  tissue,  244 
in  urine,  26,  259,  285,  300,  31s 
test  for,  316 


Nucleoproteins,  occurrence  of,  105 

Ott's  precipitation  test  for,  316 
Nylander's  reagent,  preparation  of,  28,  307 
test,  28,  307 

Obermayer's  test  for  indican,  276 
reagent,  preparation  of,  276 
Oblitine,  232 
"  Occult"  blood  in  feces,  171,  174 

tests  for,  174 
Olein,  129 
Olive  oil,  132 

emulsification  of,  133 
neutral,  preparation  of,  133 
Opalisin  in  milk,  217 
Orcin  test,  36 

Organic  physiological  constituents  of  urine,  259 
Organized  ferments,  i 
Organized  urinary  sediments,  346 
Osbome-Folin    method    for    determination     of 

total  sulphur  in  urine,  380 
Ossein,  229 

preparation  of,  229 
Osseoalbumoid,  229 
Osseomucoid,  85,  104,  229 

chemical  composition  of,  104 
Osseous  tissue,  229 

experiment  on,  229 
Ott's  precipitation  test  for  detection  of  nucleo- 

protein  in  urine,  316 
Ovalbumin,  84 
Ovoglobulin,  84 

Oxalated  plasma,  preparation  of,  197 
Oxalic  acid,  259,  279,  403 
formula  for,  279 
in  urine,  259,  279 
quantitative  determination  of,  402 
Oxaluria,  280 
Oxaluric  acid,  259,  285 
Oxamide,  90 
Oxidases,  217 
Oxyacids,  158,  163,  167 

tests  for,  167 
/?-oxybutyric  acid,  327,  397 

Black's  method  for  determination  of, 

397 
Black's  reaction  for,  327 
formula  for,  327 
Kulz's  test  for,  328 
origin  of,  327 

polariscopic  examination  for,  328 
quantitative  determination  of,  396 
Shaffer's  method  for  determination  of, 
396 
Oxyhiemoglobin,  60 

Reichert's   method    for   crystallizaticm    of, 

197 
crystalline  forms  of,  182,  i8s 
Oxymandelic  acid,  259.  284 
Oxyproline,  79 
Oxyproteic  acid,  259,  280,  337 

Paduschka-Undcrhill-Kleiner  method  for  quan- 
titative determination  of  allantoin,  401 
P.ilinitic  acid,  129,  140 

crystalline  form  of,  134 

experiments  on,  135 


INDEX. 


433 


Palmitic  acid,  formula  for,  129,  140 

preparation  of,  134 
Palmitin,  129 
Pancreatic  amylase,  138,  139.  i44 

digestion  of  dry  starch  by,  140,  145 

inulin  by,  145 
experiments  on,  144 
influence  of  bile  upon  action  of,  145 

metallic  salts  upon  action  of,  144 
most  favorable  temperature  for  action 
of,  144 
Pancreatic  digestion,  137 

general  experiments  on,  142 
products  of,  138,  142 
Pancreatic  insufficiency,   Schmidt's  nuclei  test 

for,  177 
Pancreatic  juice,  137-140 

artificial,  preparation  of,  141 
daily  excretion  of,  138 
enzymes  of,  138 
freezing-point  of,  138 
mechanism  of  secretion  of,  137 
reaction  of,  137 
solid  content  of,  138 
specific  gravity  of,  138 
Pancreatic  lipase,  129,  138,  140 
experiments  on,  145 
ethyl-butyrate  test  for,    146 
litmus-milk  test  for,  145 
Pancreatic  protease   (see  Trypsin,   p.    i) 
Pancreatic  rennin,  138,  140 

experiments  on,  146 
Papain,  10 

Para-cresol-sulphuric  acid,  259,  274 
Paradimethylamino  benzaldehyde  solution,  pre- 
paration of,  166 
Paralactic  acid,  233,  260,  286 
Paramyosinogen,  231 
Paranucleoprotagon,  244,  246 
Paraoxyphenylacetic  acid,  158,  164,  259,  282 
Paraoxyphenyl-a-amino-propionic  acid,  68 
Paraoxyphenylpropionic  acid,  158,  164,  260,  282 
Paraphenelenediamine  hydrochloride,  222 
Parasites,  171,  346,  356 
Paraxanthine,  260,  289 
Parietal  cells,  116 
Parotid  glands,  characteristics  of  saliva  secreted 

by,  51 
Pathological  constituents  of  urine,  300 
Pathological  urine,  249,  300 
constituents  of,  300 
experiments  on,  300,  337 
Pektoscope,  255 
Pentapeptides,  86 
Pentoses,  20,  35 

experiments  on,  35 
in  urine,  300,  330 
tests  for,  330 
Pepsin  (see  Gastric  Protease),  2,  9,  118 
action  of,  influence  of  bile  upon,  126 

influence  of  dilTerent  acids  upon,  83 
metallic  salts  upon,  125 
temperature  upon,  123 
conditions  essential  for  action  of,   117 
differentiation    of.    from    pepsinogen,   123 
formation  of,  117 
digestive  properties  of,  117 
28 


Pepsin,  most  favorable  acidity  for  action  of,  117 

proteolytic  action  of,  117 
Pepsin-hydrochloric  acid,  122,  125 
Pepsinogen,  5,  117,  119 

differentiation  of,  from  pepsin,  123 
formation  of,  170 
extract  of,  preparation  of,  119 
Peptic  activity,  Fuld  and  Levison's  method  for 
determination  of,  18 
Mett's  method  for  the  determination 
of,  17 
Peptic  proteolysis,  117 

products  of,  117 

relation  of,  to  tryptic  proteolysis,  118 
Peptides,  62,  64,  86,  iii,  112 
Peptone,  61,  86,  87 
ampho,  86,  87 
anti,  86,  87 

differentiation  of,  from  proteoses,  iii 
experiments  on,  112 
in  urine,  300,  314 
tests  for,  314 
separation  of,  from  proteoses,  iii 
Periodide  test  for  choline,  247 
Peroxidases,  217 
Pettenkoper's  test  for  bile  acids,  153,  320 

Mylius's  modification  of,  153, 

321 
Neukomm's    modification  of, 

153.  ?2i 
Phenaceturic  acid,  260,  286 
Phenol,  158 

tests  for,  166 
Phenolphthalein  as  indicator,  121 

preparation  of,  121 
Phenol-sulphuric  acid,  259,  274 
Phenyl-a'-amino  propionic  acid,  67 
Phenylalanine,  61,  67 
Phenyldextrosazone,  23 

crystalline  form  of,  Plate  III,  opposite  p.  23 
Phenylhydrazine,  23,  24 

acetate  solution,  preparation  of,  23 
mixture,  preparation  of,  23 
reaction,  23 

CipoUina's  modification  of,  24 
Phenyllactasazone,    crystalline    form    of,    Plate 

III,  opposite  p.  23 
Phenylmaltosazone,   crystaline    form   of,    Plate 

III,  opposite  p.  23 
Phenylpotassium  sulphate,  274 
Phosphates  in  urine,  260,  294 
detection  of,  296 
experiments  on,  296 
quantitative   determination   of,   3S3 
Phosphatides.  85,  148 
Phosphocarnic  acid,  232,  237,  260,  2S7 
Phosphoproteins,  85,  86,  105 
Phosphorized  compounds  in  urine.   260,   286 
Physiological  constituents  of  urine.  259 
Pigments  of  urine,  249,  260,  287 
Pine  wood  test  for  indole.  165 
Piria's  test  for  tyrosine,  81 
Polariscope,  use  of,  30 

in  detection  of  conjugate  glycuronates, 

329 
in  determination  of  dextrose,  31 
3-oxybutyric  acid.  328 


434 


INDEX, 


Polypeptides.  62,  64 
Polysaccharides,  21,  41 
classification  of,  21 
properties  of.  41 
Posner's  modification  of  biuret  test.  91 
Potassium  in  urine,  260.  297 
Potassium  indoxyl-sulphate    (see    Indican,    pp. 
158,  275) 
formula  for,  159,  275 
origin  of,  158,  17s 
tests  for,  275 
Potassium  iodide  test  for  albumin,  97,  312 
Primary  protein  derivatives,  85 
Primary  proteoses,  1 1 1 
Products  of  protein  hydrolysis,  61,  65 
Prolamins.  103 

classification  of,  85.  86 
Proline,  61,  78,  103,  138 

crystalline  form  of  bevo-«-  78 
crystalline  form  of  copper  salt  of,  79 
Prosecretin,  137 
Protagon,  244.  245 

preparation  of,  246 
Protamines,  classification  of,  84 
Proteans,  85,  106 
Protease,  gastric.  9 

experiments  on,  9 
pancreatic.  9 

experiments  on,  9 
vegetable,  10 
Proteases,  9 

experiments  on,  9 
Proteins,  59 

acetic    acid    and    potassium    ferro-cyanide 

test  for.  97 
Acree-Rosenheim  test  on,  91 
action  of  alkaloidal  reagents  on,  95 
action  of  metallic  salts  on,  95 

mineral    acids,    alkalies    and    organic 
acids  on,  94 
Adamkiewicz  reaction  on,  89 
Bardach's  reaction  on,  92 
biuret  test  on,  89 
chart  for  use  in  review  of,  114 
chemical  composition  of,  S9 
classification  of,  83,  84,  86 
coagulation  or  boiling  test  for,  97 
color  reactions  of,  88 
conjugated,  85,  87,  104 
decomposition  of,  60 
by  hydrolysis,  61 
by  oxidation,  61 
products  of,  61 

experiments  on,  80 
separation  of,  80 
study  of,  61,  80 
derived,  85 

formation  of  fat  from,  131 
formulas  of,  60 
Heller's  ring  test  on,  95 
importance  of,  to  life,  59 
Hopkins-Cole  reaction  on,  89 
in  urine,  300,  308 
test  for,  309 
Liebcrmann's  reaction  on,  91 
Millon's  reaction  on,  88 
molecular  weights  of,  60 


Proteins,  Posner's  reaction  on,  91 
precipitation  of,  by  alcohol,  98 
alkaloidal  reagents,  95 
metallic  salts,  95 
mineral  acids,  94 
precipitation  reactions  of,  93 
quantitative    determination    of,    in    milk, 

406 
review  of,  112 
Robert's  ring  test  on,  95 
salting-out  experiments  on,  97 
scheme  for  separation  of,  113 
simple,  84,  87 
synthesis  of,  64 
xanthoproteic  reaction  on,  89 
coagulated,  109 

biuret  test  on,  110 
formation  of,  109 
Hopkins-Cole  reaction  on,  no 
Millon's  reaction  on,  110 
solubility  of,  109,  no 
xanthoproteic  reaction  on,  no 
Protein-coagulated  enzymes,  3,  119,  215 
Proteins,  conjugated,  85,  104 
classes  of,  85,  104 
experiments    on,    191,    192,    195,   197 

199,  219 
nomenclature  of,  85,  104 
occurrence  of,  104 
Protein-cystine,  71 
Protein  derivatives,  primary,  61,  106 

secondary,  66,  in 
Proteins  of  milk.   214,  216,  217 

quantitative  determination  of,  406 
Proteolytic  enzymes  (see  Proteases,  p.  9) 
Proteolysis,  peptic,  117 

tryptic,  118 
Proteose,  61,  86,  87,  no 

V.  Aldor's  method  for  detection  of,  315 

biuret  test  on,  112 

coagulation  test  on,  112 

deutero,  86,  87,  in 

differentiation  of,  from  peptone,  in 

experiments  on,  112 

hetero,  87,  1 1 1 

in  urine,  300,  308,  314 

test  for,  314 
potassium  ferrocyanide  and  acetic  test  on, 

112 
powder,  preparation  of,  n  i 
precipitation  of,  by  nitric  acid,  112 
by  picric  acid,  112 
by  potassio  mercuric  iodide,  112 
by  trichloracetic  acid,  112 
primary,  1 1 1 
proto,  86,  87,  n  I 

Schulte's  method  for  detection  of,  314 
secondary,  1 1 1 

separation  of,  from  peptones,  in 
Protoproteose,  86,  87 
Proteoses  and  peptones,  86,  87,  1 12 
separation  of,  112 
tests  on,  112 
Protcose-peptone,  112 

Proteosc-peptone,  coagulation  test  on,  112 
experiments  on,  112 
Millon's  reaction  on,  112 


INDEX. 


435 


Proteose-peptone,    precipitation    of,    by    nitric 

acid,  112 

by  picric  acid,  112 
Prothrombin,  187,  188 
Pseudo-globulin,  178,  179,  232 
Ptomaines  and  leucomaines  in  urine,  269,  .289 
Ptyalin  (see  Salivary  amylase,  52) 
Purdy's  method  for  determination  of  dextrose, 
36s 
solution,  preparation  of,  365 
Purine  bases,  105,  399 

in  urine,     quantitative    determination     of, 
399 
Pus  casts  in  urinary  sediments,  346,  354 
Pus  cells  in  urinary  sediments,  346,  348 
Putrefaction,  indican  as  an  index  of,  158,  274 
Putrefaction  mixture,  preparation  01  a,  160 
Putrefaction  products,  158 

experiments  on,  160 

most  important,  158 

tests  for,  164 
Pyloric  glands,  115 
Pyrocatechin-sulphuric  acid,  260,  274 
a-pyrrolidine-carboxylic  acid  (see  Prolin,  p.  78) 

Qualitative  analysis  of  the  products  of  salivary 
digestion,  58 
stomach  contents,  126 
Quantitative  analysis  of  blood,  409 

of  gastric  juice,  407 

of  milk,  404 

of  urine,  361 
Quantitative     determination     of     ammonia    in 
urine,  375 

amylolytic  activity,  16 

acetone  in  urine,  394 

acetone  and  diacetic  in  urine,  391 

acidity  of  urine,  398 

allantoin  in  urine,  401 

ash  of  milk,  40s 

caseinogen  of  milk,  406 

chlorides  in  urine,  388 

creatine  in  urine,  383 

creatinine,  386 

dextrose  in  urine,  362 

diacetic  acid  in  urine,  395 

fat  in  milk,  404 

hippuric  acid  in  urine,  377 

indican  in  urine,  397 

lactalbumin  in  milk,  406 

lactose  in  milk,  406 

nitrogen  in  urine,  375 

oxalic  acid  in  urine,  402 

/J-oxybutyric  acid  in  urine,  396 

peptic  activity,  17 

phosphorus  in  urine,  383 

protein  in  milk,  406 

protein  in  urine,  361 

purine  bases  in  urine,  399 

sulphur  in  urine,  378 

total  solids  in  milk,  405 

total  solids  in  urine,  402 

tryptic  activity,  19 

urea  in  urine,  369 

uric  acid  in  urine,  367 
Quevenne  lactometer,  determination  of  specific 
gravity  of  milk  by,  404 


Raffinose,  21,  41 
Rancid  fat,  130 

Raw  and  heated  milk  tests,  217 
Reaction  of  the  urine,  251,  295 
Reduced  alkali-haematin,  202 
Reduced  haemoglobin,  199 
Reductases,  217 

Reichert's  method    for   crystallization    of    oxy- 
hemoglobin, 197 
Remont's  method  for  detection  of  salicylic  acid 

and  salicylates,  222 
Rennin,  gastric,  118 

action  of,  upon  caseinogen,  118 

experiments  on,  125,  127 

influence  of,  upon  milk,  118,  125 

in  gastric  juice,  absence  of,  118 

nature  of  action  of,  iiS 

occurrence  of,  118 
Rennin,  pancreatic,  138,  140 
experiments  on,  146 
Reticulin,  104 

Reversibility  of  enzynae  action,  6,  53 
RejTiolds-Gunning  test  for  acetone,  324 
Rhamnose,  20,  36 
Ricin,  10,  192 
Riegler's  reaction,  24,  301 
Ring  test  for  urobilin,  289 

Roaf's  method  for  crystallizing  hippuric  acid,  278 
Robin's  reaction  for  urorosein,  336 
Robert's  ring  test  for  protein,  95,  310 

reagent,  preparation  of,  95,  310 
Rosenheim's  bismuth  test  for  choline,  24S 
Rosenheim's  periodide  test  for  choline,  247 
Rossi's  reaction  for  indican,  276 
Rubner's  test  for  lactose  in  urine,  332 

Saccharide  group,  21 
Saccharose  (see  Sucrose) 
SahU's  desmoid  reaction,  124 
Saliva,  52 

alkalinity  of,  53 

amount  of,  53 

bacteria  in,  54 

biuret  test  on,  55 

calcium  in,  53 

chlorides  in,  56 

constituents  of,  53 

digestion  of  dry  starch  by,  57 

digestion  of  inulin  by,  57 

digestion  of  starch  paste  by,  54,  37 

enzymes  contained  in,  53,  54 

excretion  of  potassium  iodide  in,  59 

inorganic  matter  in,  tests  for,  56 

Millon's  reaction  on,  54 

mucin  from,  preparation  of,  55 

nitrites  in,  test  for,  56 

phosphates  in,  test  for,  56 

potassium  thiocyanate  in,  53 

reaction  of,  53,  55 

secretion  of,  52 

specific  gravity  of.  53,  55 

sulphates  in,  test  for,  56 

thiocyanates  in,  53 

tests  for,  56 
Salivary  amylase,  i,  53,  116 

acti\'ity  of.  in  stomach,  54,  116 
inhibition  of  activity  of,  54 


436 


INDEX. 


Salivary  amylase,  nature  of  action  of,  53 

products  of  action  of,  54 
Salivarj-  digestion,  52 

influence  of  acids  and  alkalis  on,  54,  5S 
dilution  on,  58 
metallic  salts  on,  58 
temperature  on,  57 
nature  of  action  of  acids  and  alkalis 

on,  58 
qualitative  analysis  of  products  of,  59 
Salivarj-  digestion  in  stomach,  54,  116 
Salivarj-  glands.  52 
Salivarj-  stimuli.  52 

Salkowski-Autenrieth-Barth  method  for  deter- 
mination of  oxalic  acid  in  urine,  402 
Salkowski's  method  for  determination  of  purine 

bases,  401 
Salkowski-Schippers  reaction  for  bile  pig    ents, 

152.  302 
Salkowski's  test  for  cholesterol,  155,  248 

for  creatinine,  274 
Salmine.  85,  86 

Salted  plasma,  preparation  of,  197 
Salting-out   experiments  on  proteins,   93,   97 
Sarcolactic  acid,  233 

Scallops,   preparation  of  glycogen  from,   240 
Schalli Jew's  method  for  preparation  of  hamin, 

195 
Scheme  for  analj'sis  of  biliarj-  calculi,   154 
bone  ash.  230 
stomach  contents,  127 
urinary  calculi,  35 ) 
separation  of  carbohydrates,  50 
of  proteins,  1 13 
Scherer's  coagulation  method  for  determination 

of  albumin  in  urine,  361 
Schiflf's  reaction  for  cholesterol,  155,  248 

for  uric  acid.  270 
Schiff's  reagent,  preparation  of,  155,  248 
Schmidt's    nuclei     test  for    pancreatic     insuffi- 
ciency, 177 
Schmidt's  test  for  hydrobilirubin,  175 
Schulte's  method   for  detection  of   proteose  in 

urine,  314 
Schumm's  modification  of  the  guaiac  test,  192 
Schutz's  law,  statement  of,  7,  18 
Schweitzer's  reagent,  action  of,  on  cellulose,  49 

preparation  of,  49 
Scleroprotcins,  83  (see  Albuminoids) 
Scombrine,  85 
Scombrone,  84,  86 
Secondary  protein  derivatives,  86 
Secondary  proteoses.  1 1 1 
Secretin,  137 
ScIiwanofT's  reaction,  34,  334 

reagent,  ijreparation  of,  34,  334 
Separation  of  feces,  importance  of,  in  nutrition 

and  metabolism  experiments,  1 70 
Serine,  61,  67 

crystalline  form  of,  67 
formula  for,  67 
Serum  albumin,  84,  87.  178.  300,  30K 
in  urine,  300,  308 
test  for,  309 
Scrum  gloViulin,  84,  178,  300,  313 
in  urine,  300,  313 
test  for,  3 1 3 


Shaffer's  method  for  determination   of  ,3-ox\-- 

but\-ric  acid,  396 
Sherman's  compressed   oxj-gen   method  for  de- 
termination of  total  sulphur  in  urine,  383 
Sherrington's  solution,  preparation  of,  209 
Silicates  in  urine,  2O0,  299 
Skatole,  158,  166 
tests  for,  166 
Skatole-carbonic  acid,  163 

test  for,  167 
Smith's  test  for  bile  pigments,  152,  320 
Soap,  salting-ovit  of,  134 
Soaponification,  129 

of  lard,  135 
Sodium  and  potassium  in  urine,  260,  297 
Sodiuni  alizarin  sulphonate  as  indicator,  122 

preparation  of,  122 
Sodium  chloride,  crj-stalline  form,  196 
Sodium  chloride  in  urine,  260,  293,  389 
Sodium  hydroxide   and    potassium    nitrate   fu- 
sion method  for  determination    of  total  sul- 
phur and  phosphorus  in  urine,  381,  384 
Sodium  hypobromite  solution,  preparation  of, 

370 
Sodium  sulv^hide  solution,  preparation  of,  400 
Solera's  reaction  for  detection  of  thiocyanate  in 
saliva,  56 
test  paper,  preparation  of,  56 
Soluble  starch,  8,  53 

Soxhlet  apparatus  for  extraction  of  fat,  404 
Soxhlet    lactometer,    determination    of    specific 

gravity  of  milk  by,  404 
Specificity  of  enzyme  action,  6 
Spectroscope,  use  of   in  detection  of  blood,  318 
Spermatozoa  in  urinary  sediments,  351,  355 
microscopical  appearance  of  human,  355 
Spiegler's  ring  test  for  protein,  96,  311 

reagent,  preparation  of,  96,  311 
Sprigg's    method    for    determination    of    peiitic 

activity,  17 
Standard  ammonium  thiocj'anate  soUUion,  pre- 
paration of,  391 
argentic    nitrate    solution,    preparation    of, 

390 
uranium   acetate  solution,   preparation   of, 
3«3 
Starch,  21,42 

action  of  alcohol  on  iodiilc  of,  45 
action  of  alkali  on  iodide  of,  45 

heat  on  iodide  of,  45 
dry,  digestion    of,  by    pancreatic  amylase, 

140,  14S 
dry,  digestion  of,  by  salivary  amylase,  57 
experiments  on,  43 
iodine  test  for,  43 
microscoi)ical  characteristics  of,  43 
microscopical  examination  of,  43 
potato,  preparation  of,  43 
soluble,  S3 
solubihty  of,  43 
various  forms  of.  44 
Starch  group,  2  1 

Starch  paste,  action  of  tannic  acid  on,  45 
(lifTusihility  of,  45 

digestion    of,    Viy    pancreatic   amylase, 
139,  144 
by  salivary  amylase    <^^.  i;7 


INDEX. 


437 


Starch  paste,  Fehling's  test  on,  45 
hydrolysis  of,  45 
iodic  acid  paper,  57 
preparation  of,  43 
Steapsin  (see  Pancreatic  lipase,  129) 
Stearic  acid,  245 
Stearin,  130 

Stellar  phosphate,  220,  340 
Stercobilin,  169 
Stokes'  reagent,  action  of,  199,  202 

preparation  of,  199 
Stomach,  motor  and  functional  activities  of,  125 
Stomach  contents,  lactic  acid  in  tests  for,   126 

qualitative  analysis  of,  126 
Stone-cystine,  71 
Sturine,  85 
Sublingual     glands,     characteristics     of     saliva 

secreted  by,  52 
Submaxillary   glands,    characteristics   of   saliva 

secreted  by,  52 
Substrate,  2,  6 
Sucrase,  11,  141 

experiments  on,  11 
vegetable,  11 
Sucrose,  21,  40 

experiments  on,  41 
inversion  of,  40 
production  of  alcohol  from,  41 
structure  of,  41 
Sulphanilic  acid,  337 
Sulphates  in  saliva,  test  for,  56 
Sulphates  in  urine,  260,  291 
experiments  on'  292 
ethereal,  274,  291 

quantitative  determination  of ,  380 
inorganic,  291 

quantitative     determination     of, 
379 
total,    quantitative    determination    of, 
378 
Sulphocyanides  (see  Thiocyanates,  53) 
Sulphur  in  protein,  100 

loosely  combined,  test  for,  100 
in  urine,  quantitative  determination  of, 

378 
in  acid,  100 
lead  blackening,  100 
mercaptan,  100 
neutral,  291 
oxidized,  100 
unoxidized,  100 
Suspension  of  manganese  dioxide,  400 

Tallow  bayberry,  saponification  of,  133 
Tallquist's  haemoglobin  scale,  determination  of 

hemoglobin  by,  208 
Tannic  acid,  influence  of,  on  dextrin,  48 

on  starch,  45 
Tannin  test  for  carbon  monoxide  haemoglobin , 

20I 

Tanret's  reagent,  preparation  of,  96,  312 
Tanret's  test,  96 
Tartar,  formation  of,  52 
Taurine,  148,  236 

derivatives,  260 

formula  for,  148,  236 

preparation  of,  155 


Taurocholic  acid,  148 

group,  148 
Taylor's  test  for  acetone,  324 
Teichmann's    crystals,    form    of     (see    Haemin 

crystals,  p.  194) 
Tendomucoid,  85,  104,  224 
biuret  test  on,  225 
chemical  composition  of,  104 
hydrolysis  of,  225 

loosely  combined  sulphur  in,  test  for,   225 
preparation  of,  224 
solubility  of,  225 
Tetrapeptides,  86,  87 

Thiocyanates  in  saliva,  significance  of,   53 
ferric  chloride  test  for,  55 
Solera's  reaction  for,  55 
Thiocyanates  in  urine,  259,  280 
Thiophene,  126 

Thoma-Zeiss  haemocytometer,  208 
Thrombin,  187,  188 
Thymus  histone,  84 
Thymol,  formula  for,  257 

interference   of,    in   Lieben's   acetone    test, 

324 
interference  in  Heller's  ring  test,  310 
use  of,  as  preservative,  257 
Tincture  of  iodine,  preparation  of,  418 
Tissue,  adipose,  experiments  on,  128,  230 
connective,  223 

white  fibrous,  223 

composition  of,  224 
experiments  on,  224 
yellow  elastic,  226 

composition  of,  226 
experiments  on,  227 
epithelial,  223 

experiments  on,  223 
muscular,  221 

experiments  on,  237 
nervous,  244 

experiments  on,  246 
osseous,  229 

experiment  on,  229 
Tissue  debris  in  urinary  sediments,  346,  356 
Toison's  solution,  preparation  of,  209 
ToUen's    reaction    on    conjugate    glycuronates, 
329 
galactose,  35 
arabinose,  36 
Topfer's   method   for   quantitative   analysis   of 

gastric  juice,  407 
Topfer's  reagent,  as  indicator,  120 

preparation  of,  120 
Total  solids,   of   milk,   quantitative   determina- 
tion of,  40s 
of    urine,   quantitative    determination 
of,  402 
Total   sulphur  of   urine,  quantitative   determi- 
nation of,  380-383 
phosphorus    of   urine,    quantitative    deter- 
mination of,  384 
Tri-butyrin,  214 
Trimethyl-oxyethyl-ammonium  hydroxide  (see 

Choline,  244) 
Tri-olein,  130,  214 
Tri-palmitin.  129,  140,  241 
Tri-stearin,  130,  214 


438 


INDEX. 


Trichloracetic    acid,    precipitation    of    protein 

by.  95 
Trioses,  21 
Tripeptides,  86,  8- 
Triple  phosphate,  243.  ^9'J.  339 
cr>-stalline  form  of,  296 
formation  of,  296 
Trisacchatides,  21,  42 
Trommer's  test,  26 
TropseoUn  00,  as  indicator,  121 

preparation  of,  121 
Trypsin  (see  also  Pancreatic  protease,  1).  9 
action  of,  upon  proteins,  63 
experiments  on,  142 
influence  of  alkalis  and  mineral  acids  upon, 

138 
nature  of.  138 
pure,  preparation  of,  138 
Try-psinogen,  5.  138 

activation  of,  139 
Tryptic  digestion,  137 

influence  of  bile  on,  143 

metallic  salts  on,  143 
most  favorable  reaction  for,  142 

temperature  for,  143 
products  of,  138,  142 
Trj'ptic  proteolysis,  118,  138 
Tryptophane,  61,  71,  138,  142 
bromine  water  test  for,  142 
formula  for,  7 1 

group  in  the  protein  molecule,  89 
Hopkins-Cole  reaction  for,  89 
occurrence  of,  as  a  decomposition  product 

of  protein,  61,  71 
occurrence  of,  as  an   end-product  of   pan- 
creatic digestion,  138,  142 
"Twinning"  of  oxyhtemoglobin  crystals,  186 
Tyrosine,  61,  68.  88,  138 

crystalline  form  of,  68,  70 
experiments  on,  81 
formula  for,  68 
Hoflfmann's  reaction  for,  81 
in  urinary  sediments,  339,  344 
microscopical  examination  of,  81 
Momer's  test  for,  82 
occurrence  of,  68 
Piria's  test  for,  81 
salts  of,  69 

separation  of,  from  leucine/  68,  80 
solubility  of,  81 
sublimation  of,  81 
Tyrosine-sulphuric  acid,  81 

V.  Udransky's  test  for  bile  acids,  153,  321 
Ufflelmann's  reagent,  preparation  of,  126 

reaction  for  lactic  acid,  126 
Unknown  substances  in  urine,  300,  337 
Unorganized  ferments,  i 
Uranium    acetate    method    for    determination 

of  total  phosphates  in  urine,  383 
Uracil,  105 

Wheeler-Johnson  reaction  for,  los 
Urate,  ammonium,  crystalline    form    of,    Plate 
VI,  opposite  p.  343 

Hodium,  crystalline  form  of,  343 
Urates  in  urinary  sediments,  339,  342 
Urea,  2^9,  260 


Urea,  crj-stalline  form  of.  261 

decomposition  of,  by  sodium  hypobromite, 

263,  266 
excretion  of,  261 
experiments  on,  264 
formation  of,  262 
formula  for,  260 
furfurol  test  for,  266 
isolation  of.  from  the  urine,  264 
melting-point  of,  264 
quantitative  determination  of,  369 
Urea  nitrate,  265 

crystalline  form  of.  263 
formvila  for,  263 
oxalate,  265 

crystalline  form  of,  265 
formula  for,  263 
Urethral  filaments  in  urinary  sediments,    346, 

355 
Uric  acid,  28,  232,  237,  259,  266,  367 
crystalline  form  of  pure,  269 
endogenous,  267 
exogenous,  267 
experiments  on,  269 
formula  for,  267 
in  leukremia,  269 
in  urinary  sediments,  341 

crystalline  form  of  Plate   V,   op- 
posite p.  267,  342 
isolation  of,  from  the  urine.  269 
Moreigne's  reaction  for,  270 
murexide  test  for,  269 
origin  of,  267 
quantitative  determination  of,  367 

Folin-SchalTer     method     for, 

367 
Heintz  method  for,  368 
Kriiger  and  Schmidt's  meth- 
od for,  368 
reducing  power  of,  27 
SchifT's  reaction  for,  270 
Uricolytic  enzymes,  3,  14 

experiments  on,  14 
Urinary  calculi,  357 

calcium  carbonate  in,  358 

oxalate  in,  358 
cholesterol  in,  360 
compound,  357 
cystine  in,  358 
fibrin  in,  358 
indigo  in,  360 
hposphates  in,  358 

scheme  for  chemical  analy.sis  of,  359 
simple,  357 

uric  acid  and  urates  in,  358 
urostealiths  in,  358 
xanthine  in,  358 
Urinary  concrements    (see    Urinary   calculi,    p. 

357) 
Urinary  concretion  (see  Urinary  calculi,  p.  357) 
Urination,  frequency  of,  251 
Urinary  sediments,  338 

ammonium      magnesium       phosphate 

in,  339 
animal  parasites  in,  346,  356 
calcium  carbonate  in,  340 
oxalate  in,  339 


INDEX. 


439 


Urinary  sediments,  calcium  phosphatejn,  340 
sulphate  in,  341 

casts  in,  346,  349 

cholesterol  in,  344 

collection  of,  338 

cylindroids  in,  354 

cystine  in,  343 

epithelial  cells  in,  346,  347 

erythrocytes  in,  346,  355 

fibrin  in,  346,  356 

foreign  substances  in,  346,  336 

haematoidin  and  bilirubin  in,  339,  345 

hippuric  acid  in,  344 

indigo  in,  339,  346 

leucine  and  tyrosine  in,  339,  344 

magnesium  phosphate  in,  339,  345 

melanin  in,  339,  346 

micro-organisms  in,  346,  356 

organized,  338 

pus  cells  in,  346,  338 

spermatozoa  in,  346,  355 

tissue  debris  in,  346,  356 

unorganized,  338,  346 

urates  in,  339,  342 

urethral  filaments  in,  346,  355 

uric  acid  in,  341 

xanthine  in,  339,  345 
Urine,  249—403 

acetone  in,  322 

acidity  of,  251,  295 

acid  fermentation  of,  253 

albumin  in,  300,  305 

alkaline  fermentation  of,  251 

alantoin  in,  259,  280 

ammonia  in,  252,  260,  290 

aromatic  oxyacids  in,  259,  282 

benzoic  acid  in,  259,  284 

bile  in,  319,  320 

blood  in,  300,  316 

calciuip  in,  298 

carbonates  in,  260,  298 

chlorides  in,  260,  293 

collection  of,  257 

conjugate  glycuronates  in,  300,  329 

color  of,  249 

creatinine  in,  259,  270 

dextrose  in,  300 

diacetic  acid  in,  300,  326 

electrical  conductivity  of,  256 

enzymes  in,  260,  285 

ethereal  sulphuric  acid  in,  259,  274 

fat  in,  300,  330 

fluorides  in,  260,  293,  389 

freezing-point  of,  255 

galactose  in,  300,  332 

general  characteristics  of,  249 

globulin  in,  300,  313 

Haser's  coefficient  for  solids  in,  254,  403 

hsematoporphyrin  in,  300,  331 

hippuric  acid  in,  277,  344 

hydrogen  peroxide  in,  260,  299 

inorganic     physiological     constituents     of, 

290 
inosite  in,  300,  334 
iron  in,  260,  298 
lactose  in,  300,  331 


Urine,  tevulose  in,  300,  323 
laiose  in,  300,  33s 
leucomaines  in,  260,  289 
Long's  coefficient  for  solids  in,  254,  403 
magnesium  in,  260,  298 
melanin  in,  300,  335 
neutral  sulphur  compounds  in,  280 
nitrates  in,  260,  299 
nucleoprotein  in,  259,  285,  300,  315 
odor  of,  251 

organic  physiological  constituents  of,  259 
oxalic  acid  in,  259,  279 
oxaluric  acid  in,  259,  285 
/9-oxybutyric  acid  in,  327,  396 
pathological  constituents  of,  300 
paralactic  acid  in,  232,  260,  286 
pentoses  in,  300,  330 
peptone  in,  300,  314 
phenaceturic  acid  in,  260,  286 
phosphates  in,  260,  294 
phosphorized  compounds  in,  260,  286 
physiological  constituents  of,  259 
pigments  of,  249,  260,  287 
potassium  in,  260,  297 
proteins  in,  300,  308 
proteoses  in,  300,  308,  314 
ptomaines  in,  260,  289 
purine  bases  in,  399 
quantitative  analysis  of,  361 
reaction  of,  251,  295 
silicates  in,  260,  299 
sodium,  260,  297 
solids  of,  254,  402 
specific  gravity  of,  253 
sulphates  in,  260,  291 
transparency  of,  250 
unknown  substances  in,  300,  337 
urea  in,  259 
uric  acid  in,  259,  266 
urorosein  in,  300,  336 
volatile  fatty  acids  in,  260,  285 
volume  of,  249 
Urobilin,  249,  260,  287 

tests  for,  288 
Urochrome,  249,  287 
Uroerythrin,  10,  249,  287,  307 
Uroferric  acid,  259,  280,  337 
Uroleucic  acid,  259,  283 
Urorosein,  300,  336 
tests  for,  336 

Valine,  73 
Vegetable  amylase,  8 

lipase,  10 

protease,  9 

sucrase,  11 
Vegetable  globulins,  84 
Vegetable  gums,  21 
Veith    lactometer,     determination    of    specific 

gravity  of  milk  by,  404 
Viscosity  test,  56 
Vitellin,  85,  86 

Volatile  fatty  acids,  158,  161,  260,  285 
Volhard-Amold   method   for   determination   of 

chlorides,  391 
Volume  of  the  urine,  249 


440 


INDEX. 


Wax  myrtle,  133 

Waxy  casts  in  urinarv'  sediments.  346,  553 

Weber's  guaiac  test  for  blood  in  feces,  174 

Weinland,  formation  of  fat  from  protein,  132 

Welker's  modified  method  for  purine  bases,  39S 

Weyl's  test  for  creatinine,  273 

Wheeler-Johnson  reaction  for  uracil  and  cyto- 
sine,  105 

White  fibrous  connective  tissue,  223 
experiments  on,  224 

Wilkinson  and  Peters'  test,  218 

Wirsing's  test  for  urobilin,  288 

Wohlgemuth's  method  for  quantitative  deter- 
mination of  amylolytic  activity,  16 

Xanthine,  232,  235,  237 

crystalline  form  of,  235 

formula  for,  337 

in  urinary  sediments,  339.  34  5 

isolation  of,  from  meat  extract,  241 

Weidel's  reaction  for,  242 


Xanthine  bases  (see  Purine  bases,  pp.  102,  399) 

Xanthine  silver  nitrate,  241 

crystalline  form  of,  242 

Xanthoproteic  reaction,  89 

Xylose,  20,  37 

orcin  reaction  on,  37 
phenylhydrazine  reaction  on,  37 
Tollens'  reaction  on,  36 

Yellow  elastic  connective  tissue,  226 
composition  of,  226 
e.xperiments  on,  227 

Zappert  slide,  212 

Zein,  103 

Zeller's  test  for  melanin,  336 

V.  Zeynek  and  Nencki's  haemin  test,  195,  317 

Zikel  pektoscope,  255 

Zymase,  preparation  of,  2 

Zymo-exciter,  5 

Zymogen,  s.  ii7 


